Maize promoter P67 and methods of use

ABSTRACT

A promoter isolated from  Zea mays , designated the P67 promoter, provides a high level of specificity for expression in developing pollen, particularly at the mid-uninucleate stage, as confirmed by RT-PCR and Northern blot analyses of RNA samples from various tissues. Compositions and methods of use of the P67 promoter are disclosed.

This application is a divisional of U.S. patent application Ser. No.11/014,071, filed Dec. 16, 2004, now U.S. Pat. No. 7,696,405, whichclaims priority to Provisional Application No. 60/530,478, filed Dec.16, 2003, and Provisional Application No. 60/591,975, filed Jul. 29,2004, all of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to compositions and methods for dominantgene suppression. Certain embodiments provide methods for preventingtransmission of transgenes in gametes. Certain embodiments comprisepairs of plants in which the phenotype of the parents is suppressed inthe progeny. Certain embodiments provide constructs and methods usefulfor generating fertile parental plants that, when crossed, generatesterile progeny plants, and methods of making and using such transgenesand plants, as well as products of such plants.

2. Background Information

Plant breeding provides a means to combine desirable traits in a singleplant variety or hybrid, including for example, disease resistance,insect resistance, drought tolerance, improved yield, and betteragronomic quality. Field crops generally are bred by pollination,including by self-pollination (selfing; selfed), in which pollen fromone flower is transferred to the same or another flower of the sameplant, or to a genetically identical plant, and cross-pollination(crossing; crossed), in which pollen from one plant is transferred to aflower of a genetically different plant.

Plants that are selfed and selected for type over many generationsbecome homozygous at almost all gene loci and produce a uniformpopulation of true breeding progeny. A cross between two differenthomozygous lines produces a uniform population of hybrid plants that canbe heterozygous at many gene loci. A cross of two plants, each of whichis heterozygous at a number of gene loci, generates hybrid plants, whichdiffer genetically and are not uniform.

Many crop plants, including, for example, maize (corn), can be bredusing self-pollination or cross-pollination techniques. Maize hasseparate male and female flowers on the same plant, located on thetassel and the ear, respectively. Natural pollination occurs in maizewhen wind blows pollen from the tassels to the silks that protrude fromthe tops of the ears. Many crop plants, including maize, are grown ashybrids, which generally exhibit greater vigor than the parental plantsfrom which they are derived. As such, it is desirable to prevent randompollination when generating hybrid plants.

Hybrid plants (F1) are generated by crossing two different inbred male(P1) and female (P2) parental plants. Hybrid plants are valued becausethey can display improved yield and vigor as compared to the parentalplants from which the hybrids are derived. In addition, hybrid (F1)plants generally have more desirable properties than progeny (F2) plantsderived from the hybrid plants. As such, hybrid plants are commerciallyimportant, and include many agricultural crops, including, for example,wheat, corn, rice, tomatoes, and melons. Hybridization of maize hasreceived particular focus since the 1930s. The production of hybridmaize involves the development of homozygous inbred male and femalelines, the crossing of these lines, and the evaluation of the crossesfor improved agronomic performance. Pedigree breeding and recurrentselection are two of the breeding methods used to develop inbred linesfrom populations. Breeding programs combine desirable traits from two ormore inbred lines, or various broad-based sources, into breeding poolsfrom which new inbred lines are developed by selfing and selecting fordesired phenotypes. These new inbreds are crossed with other inbredlines and the resultant new hybrids are evaluated to determine whichhave improved performance or other desirable traits, thus increasingcommercial value. The first generation hybrid progeny, designated F₁, ismore vigorous than its inbred parents. This hybrid vigor, or heterosis,can be manifested in many ways, including increased vegetative growthand increased seed yield.

Production of hybrid seed requires maintenance of the parental seedstocks because self-crossing of hybrid plants produces progeny (F2)that, like P1 and P2, generally exhibit less desirable characteristicsthan the F1 hybrid plant. Because the parental plants generally haveless commercial value than the hybrids (F1), efforts have been made toprevent parental plants in a field from self-crossing (“selfing”), sincesuch crosses would reduce the yield of hybrid seed. Accordingly, methodshave been developed to selfing of a parental plant.

One method for controlling pollination is to use a parental populationof plants that are male sterile, thus providing the female parent.Several methods have been used for controlling male fertility,including, for example, manual or mechanical emasculation (detasseling),cytoplasmic male sterility, genetic male sterility, and the use ofgametocides. For example, parental selfing in a field can be preventedby removing the anthers or detasseling plants of the female parental(P2) population, thus removing the source of P2 pollen from the field.P2 female plants then can be pollinated with P1 pollen by hand or usingmechanical means. Hybrid maize seed generally is produced by a malesterility system incorporating manual or mechanical detasseling.Alternate strips of two maize inbreds are planted in a field, and thepollen-bearing tassels are removed from one of the inbreds (P2 female).Provided that the field is sufficiently isolated from sources of foreignmaize pollen, the ears of the detasseled inbred are fertilized only bypollen from the other inbred (P1 male); resulting seed is hybrid andforms hybrid plants. Unfortunately, this method is time- andlabor-intensive. In addition, environmental variation in plantdevelopment can result in plants producing tassels after manualdetasseling of the female parent is completed. Therefore detasselingmight not ensure complete male sterility of a female inbred plant. Inthis case, the resultant fertile female plants will successfully shedpollen and some female plants will be self-pollinated. This will resultin seed of the female inbred being harvested along with the desiredhybrid seed. Female inbred seed is not as productive as F₁ seed. Inaddition, the presence of female inbred seed can represent a germplasmsecurity risk for the company producing the hybrid. The female inbredcan also be mechanically detasseled. Mechanical detasseling isapproximately as reliable as hand detasseling, but is faster and lesscostly. However, most detasseling machines produce more damage to theplants than hand detasseling, which reduces F₁ seed yields. Thus neitherform of detasseling is presently entirely satisfactory, and a needcontinues to exist for alternative hybrid production methods that reduceproduction costs, increase production safety, and eliminateself-pollination of the female parent during the production of hybridseed.

Another method of preventing parental plant selfing is to utilizeparental plants that are male sterile or female sterile. Male fertilitygenes have been identified in a number of plants and include dominantand recessive male fertility genes. Plants that are homozygous for arecessive male fertility gene do not produce viable pollen and areuseful as female parental plants. However, a result of the female plantsbeing homozygous recessive for a male fertility gene is that they arenot capable of selfing and, therefore, a means must be provided forobtaining pollen in order to maintain the parental P2 plant line.Generally, a maintainer cell line, which is heterozygous for the malefertility gene, is generated by crossing a homozygous dominant malefertile plant with the homozygous recessive female sterile plant. Theheterozygous maintainer plants then are crossed with the homozygousrecessive male sterile plants to produce a population in which 50% ofthe progeny are male sterile. The male sterile plants are then selectedfor use in generating hybrids. As such, the method requires additionalbreeding and selection steps to obtain the male sterile plants, thusadding to the time and cost required to produce the hybrid plants.

To overcome the requirement of having to select male sterile from malefertile plants generated by crossing a maintainer plant line with afemale (male sterile) plant line, methods have been developed to obtainmale sterile plants by expressing a cytotoxic molecule in cells of themale reproductive organs of a plant. For example, a nucleic acidencoding the cytotoxic molecule can be linked to a tapetum-specificpromoter and introduced into plant cells, such that, upon expression,the toxic molecule kills anther cells, rendering the plant male sterile.As above, however, such female parental plants cannot be selfed and,therefore, require the preparation and use of a maintainer plant line,which, when crossed with the male sterile female parent restoresfertility, for example, by providing a dominant male fertility gene, orby providing a means to inactivate or otherwise inhibit the activity ofthe cytotoxic gene product (see, U.S. Pat. No. 5,977,433).

Additional methods of conferring genetic male sterility have beendescribed including, for example, generating plants with multiple mutantgenes at separate locations within the genome that confer male sterility(see, U.S. Pat. Nos. 4,654,465 and 4,727,219) or with chromosomaltranslocations (see, U.S. Pat. Nos. 3,861,709 and 3,710,511). Anothermethod of conferring genetic male sterility includes identifying a genethat is required for male fertility; silencing the endogenous gene,generating a transgene comprising an inducible promoter operably linkedto the coding sequence of the male fertility gene, and inserting thetransgene back into the plant, thus generating a plant that is malesterile in the absence of the inducing agent, and can be restored tomale fertile by exposing the plant to the inducing agent (see, U.S. Pat.No. 5,432,068).

While the previously described methods of obtaining and maintaininghybrid plant lines have been useful for plant breeding and agriculturalpurposes, they require numerous steps and/or additional lines formaintaining male sterile or female sterile plant populations in order toobtain the hybrid plants. Such requirements contribute to increasedcosts for growing the hybrid plants and, consequently, increased coststo consumers. Thus, a need exists for convenient and effective methodsof producing hybrid plants, and particularly for generating parentallines that can be crossed to obtain hybrid plants.

A reliable system of genetic male sterility would provide a number ofadvantages over other systems. The laborious detasseling process can beavoided in some genotypes by using cytoplasmic male-sterile (CMS)inbreds. In the absence of a fertility restorer gene, plants of a CMSinbred are male sterile as a result of cytoplasmic (non-nuclear) genomefactors. Thus, this CMS characteristic is inherited exclusively throughthe female parent in maize plants, since only the female providescytoplasm to the fertilized seed. CMS plants are fertilized with pollenfrom another inbred that is not male-sterile. Pollen from the secondinbred may or may not contribute genes that make the hybrid plantsmale-fertile. Usually seed from detasseled normal maize and CMS-producedseed of the same hybrid must be blended to insure that adequate pollenloads are available for fertilization when the hybrid plants are grownand to insure cytoplasmic diversity.

Another type of genetic sterility is disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar, et al. However, this form of geneticmale sterility requires maintenance of multiple mutant genes at separatelocations within the genome and requires a complex marker system totrack the genes, making this system inconvenient. Patterson described agenetic system of chromosomal translocations, which can be effective,but is also very complex. (See, U.S. Pat. Nos. 3,861,709 and 3,710,511).

Many other attempts have been made to address the drawbacks of existingsterility systems. For example, Fabijanski, et al., developed severalmethods of causing male sterility in plants (see, EPO 89/3010153.8Publication Number 329,308 and PCT Application Number PCT/CA90/00037published as WO 90/08828). One method includes delivering into the planta gene encoding a cytotoxic substance that is expressed using a maletissue specific promoter. Another involves an antisense system in whicha gene critical to fertility is identified and an antisense construct tothe gene inserted in the plant. Mariani, et al. also shows severalcytotoxic antisense systems. See, EP Number 89/401,194. Still othersystems use “repressor” genes that inhibit the expression of other genescritical to male fertility. See, WO 90/08829.

A still further improvement of this system is one described at U.S. Pat.No. 5,478,369 in which a method of imparting controllable male sterilityis achieved by silencing a gene native to the plant that is critical formale fertility and further introducing a functional copy of the malefertility gene under the control of an inducible promoter which controlsexpression of the gene. The plant is thus constitutively sterile,becoming fertile only when the promoter is induced, allowing forexpression of the male fertility gene.

In a number of circumstances, a particular plant trait is expressed bymaintenance of a homozygous recessive condition. Difficulties arise inmaintaining the homozygous condition when a transgenic restoration genemust be used for maintenance. For example, the MS45 gene in maize (U.S.Pat. No. 5,478,369) has been shown to be critical to male fertility.Plants heterozygous or hemizygous for the dominant MS45 allele are fullyfertile due to the sporophytic nature of the MS45 fertility trait. Anatural mutation in the MS45 gene, designated ms45, imparts a malesterility phenotype to plants when this mutant allele is in thehomozygous state. This sterility can be reversed (i.e., fertilityrestored) when the non-mutant form of the gene is introduced into theplant, either through normal crossing or transgenic complementationmethods. However, restoration of fertility by crossing removes thedesired homozygous recessive condition, and both methods restore fullmale fertility and prevent maintenance of pure male sterile maternallines. The same concerns arise when controlling female fertility of theplant, where a homozygous recessive female must be maintained bycrossing with a plant containing a restoration gene. Therefore there isconsiderable value not only in controlling the expression of restorationgenes in a genetic recessive line, but also in controlling thetransmission of the restoring genes to progeny during the hybridproduction process.

SUMMARY OF THE INVENTION

The present invention is based on the determination that the genotype ofan organism (e.g., a plant or mammal) can be modified to containdominant suppressor alleles or transgene constructs that reduce, but notablate, the activity of a gene, wherein the phenotype of the organism isnot substantially affected. For example, plants can contain dominantsuppressor alleles and/or transgene constructs that suppress theactivity of a plant male fertility gene, without rendering the plantmale sterile, or can contain dominant suppressor alleles and/ortransgene constructs that suppress the activity of a gene required forviability, without killing the plant. Further, pairs of such plantshaving selected genotypes comprising the dominant suppressor alleles ortransgene constructs can be crossed to produce progeny that exhibit thephenotypic change (e.g., male sterility). Progeny of plants comprisingsuppressed male fertility genes, for example, can be useful as femalesin hybrid plant production.

Accordingly, in one embodiment, the present invention relates to abreeding pair of plants, wherein the plants comprising the breeding pairare fertile (i.e., male fertile and female fertile), and wherein sterileprogeny (e.g., male sterile progeny) are produced by crossing thebreeding pair of plants. A breeding pair of plants of the invention caninclude, for example, a first plant having an inactivated firstendogenous fertility gene, wherein the first plant is fertile; and asecond plant having an inactivated second endogenous fertility gene,wherein the second plant is fertile. Such a breeding pair is furthercharacterized in that, if the first endogenous fertility gene is a malefertility gene, then the second endogenous fertility gene also is a malefertility gene, and, similarly, if the first endogenous fertility geneis a female fertility gene, then the second endogenous fertility genealso is a female fertility gene.

In a breeding pair of plants of the invention, the first endogenousfertility gene and the second endogenous fertility gene can encode geneproducts that are present in a single pathway involved in determiningfertility of a plant, or the first endogenous fertility gene and thesecond endogenous fertility gene can encode gene products that are inseparate but convergent pathways. In either case, the presence of asingle inactivated fertility gene in a plant does not substantiallyaffect fertility of the plant, or plants derived therefrom, except thatwhen a first and second plant as defined herein are crossed, theinactivation of both a first and a second fertility gene in progenyplants results in the progeny plants being sterile (i.e., male sterileor female sterile).

The inactivated fertility gene can be inactivated due, for example, to amutation (e.g., deletion, substitution, or insertion of one or morenucleotides in the coding or non-coding sequence that reduces orinhibits expression of the fertility gene), including, for example,knock out of the gene (e.g., by a homologous recombination event),preferably in both alleles of the fertility gene. The inactivatedfertility gene also can be inactivated due, for example, to expressionof a gene product such as a transgene product (e.g., an RNA or anencoded polypeptide) in cells of the plant in which the gene normally isexpressed, or in progenitor cells, wherein the gene product reduces orinhibits expression of the endogenous fertility gene. Further, in abreeding pair of plants of the invention, the first endogenous fertilitygene of the first plant and the second endogenous fertility gene of thesecond plant can be inactivated in the same or different ways. Forexample, the first endogenous fertility gene can be inactivated due to amutation, and the second endogenous fertility gene can be inactivateddue to expression of a transgene product (e.g., a hairpin RNA comprisinga nucleotide sequence of the promoter of the second fertility gene).

In various embodiments, the breeding pair can include a first plant, inwhich the first endogenous fertility gene is inactivated by a mutation,and a second plant having a second endogenous fertility gene inactivatedin a manner other than a mutation; or can include a first plant in whichthe first endogenous fertility gene is inactivated by a mutation, and asecond plant in which the second endogenous fertility gene isinactivated by a mutation; or can include a first plant having a firstendogenous fertility gene inactivated in a manner other than a mutation,and a second plant in which the second endogenous fertility gene isinactivated in a manner other than by a mutation. In aspects of thisembodiment, the first or second endogenous fertility gene of the firstor second plant is inactivated by knockout of the first or secondfertility gene, respectively; or the first or second endogenousfertility gene of the first or second plant is inactivated by mutationof the promoter of the first or second fertility gene, respectively. Infurther aspects, the first and second endogenous fertility genes of thefirst and second plants are inactivated by knockout of the first andsecond fertility genes, respectively; or the first and second endogenousfertility genes of the first and second plants are inactivated bymutation of the promoter of the first and second fertility genes,respectively.

In other embodiments, in a breeding pair of plants of the invention, thefirst endogenous fertility gene is inactivated due to expression in thefirst plant of a first exogenous nucleic acid molecule comprising apromoter operably linked to a nucleotide sequence encoding a firsthairpin (hp) ribonucleic acid (RNA) molecule (hpRNA), wherein the firsthpRNA comprises a nucleotide sequence of the first endogenous fertilitygene promoter, and wherein, upon expression, the first hpRNA suppressesexpression of the first endogenous fertility gene; or the secondendogenous fertility gene is inactivated due to expression in the secondplant of a second exogenous nucleic acid molecule comprising a promoteroperably linked to a nucleotide sequence encoding a second hpRNA,wherein the second hpRNA comprises a nucleotide sequence of the secondendogenous fertility gene promoter, and wherein, upon expression, thesecond hpRNA suppresses expression of the second endogenous fertilitygene; or both the first endogenous fertility gene and second endogenousfertility gene are inactivated due to expression in the first plant andsecond plant of a first hpRNA and a second hpRNA, respectively, havingthe above-described characteristics. In aspects of this embodiment, thefirst exogenous nucleic acid molecule, when present, is stablyintegrated in the genome of cells of the first plant; or the secondexogenous nucleic acid molecule, when present is stably integrated inthe genome of cells of the second plant; or both the first exogenousnucleic acid molecule, when present, and the second exogenous nucleicacid molecule, when present, are stably integrated in the genome ofcells of the first plant and second plant, respectively.

Where a first and/or second endogenous fertility gene is inactivated dueto expression in a first and/or second plant, respectively, of anexogenous nucleic acid molecule comprising a promoter operably linked toa nucleotide sequence encoding an hpRNA, the promoter can be anypromoter that is active in plant cells, for example, a constitutivelyactive promoter, (e.g., an ubiquitin promoter), a tissue specificpromoter, particularly a reproductive tissue promoter (e.g., an antherspecific promoter such as a tapetum specific promoter), an induciblepromoter, or a developmental or stage specific promoter. The fertilitygene that is inactivated can be a male fertility gene or a femalefertility gene, provided that, if a male fertility gene is inactivatedin a first plant of a breeding pair (i.e., a first endogenous malefertility gene), the second plant of the breeding pair has aninactivated male fertility gene that is different from the firstendogenous male fertility gene; and, conversely, if a female fertilitygene is inactivated in a first plant of a breeding pair (i.e., a firstendogenous female fertility gene), the second plant of the breeding pairhas an inactivated female fertility gene that is different from thefirst endogenous female fertility gene. Further, the inactivation of afirst or second endogenous fertility gene, alone, does not render aplant sterile, whereas a cross of a first plant having the firstinactivated fertility gene and a second plant having the secondinactivated fertility gene generates progeny that are sterile.

In another embodiment, the present invention relates to a breeding pairof transgenic plants, which includes a first fertile transgenic planthaving integrated in its genome a first exogenous nucleic acid moleculecomprising a promoter operably linked to a nucleotide sequence encodinga first hpRNA, wherein the first hpRNA comprises a nucleotide sequencefrom a first endogenous fertility gene promoter, and wherein, uponexpression, the first hpRNA suppresses expression of the firstendogenous fertility gene; and a second fertile transgenic plant havingintegrated in its genome a second exogenous nucleic acid moleculecomprising a promoter operably linked to a nucleotide sequence encodinga second hpRNA, wherein the second hpRNA comprises a nucleotide sequencefrom a second endogenous fertility gene promoter, wherein the secondendogenous fertility gene is different from the first endogenousfertility gene, and wherein, upon expression, the second hpRNAsuppresses expression of the second endogenous fertility gene. Asdisclosed herein, the first endogenous gene is different from the secondendogenous gene and, further if, in a breeding pair of plants, the firstendogenous fertility gene of the first plant is a male fertility gene,then the second endogenous fertility gene of the second plant of thebreeding pair also is a male fertility gene; whereas if the firstendogenous fertility gene of the first plant is a female fertility gene,then the second endogenous fertility gene of the second plant also is afemale fertility gene.

In certain embodiments, in an exogenous nucleic acid molecule containedin a first or second transgenic plant of a breeding pair of plants ofthe invention, the nucleotide sequence encoding the first or secondhpRNA, respectively, is such that it includes the sequence of thepromoter of the fertility gene that is to be inactivated, particularlyan inverted repeat of the promoter sequence such that, upon expression,self-hybridization of the RNA results in formation of the hpRNA. Assuch, the nucleotide sequence, when expressed in a cell, forms a hairpinRNA molecule (i.e., an hpRNA), which suppresses (i.e., reduces orinhibits) expression of the endogenous fertility gene from itsendogenous promoter.

The promoter, which is operably linked to the nucleotide sequenceencoding the hpRNA in an exogenous nucleic acid molecule contained in afirst or second transgenic plant of a breeding pair, can be any promoterthat is active in plant cells, particularly a promoter that is active(or can be activated) in reproductive tissues of a plant (e.g., stamensor ovaries). As such, the promoter can be, for example, a constitutivelyactive promoter, an inducible promoter, a tissue-specific promoter, or adevelopmental stage specific promoter. Also, the promoter of the firstexogenous nucleic acid molecule can be the same as or different from thepromoter of the second exogenous nucleic acid molecule.

In general, a promoter is selected based, for example, on whetherendogenous fertility genes to be inhibited are male fertility genes orfemale fertility genes. Thus, where the endogenous genes to be inhibitedare male fertility genes (e.g., a BS7 gene and an SB200 gene), thepromoter can be a stamen specific and/or pollen specific promoter suchas an MS45 gene promoter (U.S. Pat. No. 6,037,523), a 5126 gene promoter(U.S. Pat. No. 5,837,851), a BS7 gene promoter (WO 02/063021), an SB200gene promoter (WO 02/26789), a TA29 gene promoter (Nature 347:737(1990)), a PG47 gene promoter (U.S. Pat. No. 5,412,085; U.S. Pat. No.5,545,546; Plant J 3(2):261-271 (1993)), an SGB6 gene promoter (U.S.Pat. No. 5,470,359) a G9 gene promoter (U.S. Pat. Nos. 5,837,850 and5,589,610), or the like, such that the hpRNA is expressed in antherand/or pollen or in tissues that give rise to anther cells and/orpollen, thereby reducing or inhibiting expression of the endogenous malefertility genes (i.e., inactivating the endogenous male fertilitygenes). In comparison, where the endogenous genes to be inhibited arefemale fertility genes, the promoter can be an ovary specific promoter,for example. However, as disclosed herein, any promoter can be used thatdirects expression of the hpRNA in the reproductive tissue of interest,including, for example, a constitutively active promoter such as anubiquitin promoter, which generally effects transcription in most or allplant cells.

The present invention also provides cells of a first plant or of asecond plant or of both a first plant and a second plant of a breedingpair of plants of the invention. In addition, seeds of the first plantand/or second plant are provided, as are cuttings of the first and/orsecond plant.

The present invention further relates to a transgenic non-human organismthat is homozygous recessive for a recessive genotype, wherein thetransgenic organism contains an expressible first exogenous nucleic acidmolecule comprising a first promoter operably linked to a polynucleotideencoding a restorer gene, the expression of which restores the phenotypethat is otherwise absent due to the homozygous recessive genotype, and asecond exogenous nucleic acid molecule encoding an hpRNA. The transgenicnon-human organism can be any non-human organism that has a diploid (orgreater) genome, including, for example, mammals, birds, reptiles,amphibians, or plants.

In one embodiment, the second expressible exogenous nucleic acidmolecule of a transgenic plant of the invention encodes an hpRNAspecific for the first promoter, which drives expression of the restorergene. In one aspect of this embodiment, the second expressible exogenousnucleic acid molecule further comprises a second promoter operablylinked to the nucleotide sequence encoding the hpRNA. The secondpromoter generally is different from the first promoter (of the firstexpressible exogenous nucleic acid molecule), and can be, for example, aconstitutive promoter, an inducible promoter, a tissue specificpromoter, or a developmental stage specific promoter, such that thehpRNA can be expressed in the transgenic organism in a constitutivemanner, an inducible manner, a tissue specific manner, or at aparticular stage of development. In another embodiment, the secondexpressible exogenous nucleic acid molecule of a transgenic plant of theinvention encodes an hpRNA specific for a promoter other than the firstpromoter which drives expression of the restorer gene of the firstexpressible exogenous nucleic acid molecule.

A transgenic non-human organism of the invention is exemplified hereinby a transgenic plant that is homozygous recessive for a recessivesterile genotype (e.g., homozygous recessive for the ms45 gene, which isa male fertility gene), and that contains (a) a first expressibletransgene comprising a first promoter operably linked to a nucleotidesequence encoding a restorer gene, which, upon expression, restoresfertility to the transgenic plant (e.g., transgene comprising an MS45coding sequence), and (b) a second expressible transgene encoding anhpRNA, which, upon expression, suppresses expression by a secondpromoter, which is different from the first promoter. In one embodiment,the first promoter is a constitutive or developmentally regulatedpromoter, wherein the fertility restorer gene is expressed in thetransgenic plant, and the transgenic plant is fertile. In anotherembodiment, the first promoter is an inducible promoter, wherein, uponcontact of the transgenic plant with an appropriate inducing agent,expression of the fertility restorer gene is induced, rendering thetransgenic plant fertile.

In another embodiment, the present invention also relates to a breedingpair of transgenic non-human organisms, including a first transgenicorganism and second transgenic organism each of which is homozygousrecessive for the same recessive genotype. The breeding pair is furthercharacterized in that the first transgenic organism contains anexpressible first exogenous nucleic acid molecule comprising a firstpromoter operably linked to a nucleotide sequence encoding a restorergene, the expression of which restores the phenotype that is otherwiseabsent due to the homozygous recessive genotype, and a secondexpressible exogenous nucleic acid molecule that encodes an hpRNAspecific for a second promoter, which is different from the firstpromoter. The second transgenic organism contains an expressible thirdexogenous nucleic acid molecule comprising the second promoter operablylinked to a nucleotide sequence encoding a restorer gene, the expressionof which restores the phenotype that is otherwise absent due to thehomozygous recessive genotype, and a fourth expressible exogenousnucleic acid molecule that encodes an hpRNA specific for the firstpromoter. The first and second transgenic non-human organism are furthercharacterized in that, when bred with each other, progeny are producedin which the second hpRNA inhibits expression of the restorer gene ofthe first transgene and the first hpRNA inhibits expression of therestorer gene of the third transgene, such that the progeny exhibit therecessive phenotype of the homozygous recessive genotype.

A breeding pair of transgenic non-human organisms of the invention isexemplified by a breeding pair of transgenic plants, as follows.

The first plant of the pair is a fertile transgenic plant having ahomozygous recessive sterile genotype, having integrated in its genome afirst exogenous nucleic acid molecule comprising a nucleotide sequenceencoding a fertility restorer gene operably linked to a heterologousfirst promoter, wherein expression of the restorer gene restoresfertility to the first transgenic plant; and a second exogenous nucleicacid molecule comprising a first hpRNA, wherein the first hpRNAcomprises a nucleotide sequence of a second promoter, and wherein, uponexpression, the first hpRNA suppresses expression from the secondpromoter, which is different from the first promoter.

The second transgenic plant of the pair has the same homozygousrecessive sterile genotype as the first transgenic plant, and hasintegrated in its genome a third exogenous nucleic acid molecule, whichcomprises a nucleotide sequence encoding the fertility restorer geneoperably linked to the second promoter, which is heterologous to thefertility restorer gene, wherein expression of the restorer generestores fertility to the second transgenic plant; and a fourthexogenous nucleic acid molecule comprising a second hpRNA, wherein thesecond hpRNA comprises a nucleotide sequence of the heterologous firstpromoter, and wherein, upon expression, the second hpRNA suppressesexpression of the first exogenous nucleic acid molecule comprising theheterologous first promoter.

As disclosed herein, in progeny of a cross of the first and secondtransgenic plants, the second hpRNA suppresses expression of the firstexogenous nucleic acid molecule, including the fertility restorer genecontained therein, and the first hpRNA suppresses expression of thethird exogenous nucleic acid molecule, including the fertility restorergene contained therein. As such, the progeny are sterile, for example,female sterile. A breeding pair of transgenic plants of the inventioncan be homozygous recessive for male fertility genes (i.e., malesterile, except upon expression of the fertility restorer gene), or canbe homozygous recessive for female fertility genes (i.e., femalesterile, except upon expression of the fertility restorer gene).

In one aspect, a breeding pair of transgenic plants of the inventionincludes a first transgenic plant, which is homozygous recessive forms45, wherein the first exogenous nucleic acid molecule comprises anucleotide sequence encoding MS45 operably linked to a 5126 genepromoter, and the second exogenous nucleic acid molecule comprises afirst hpRNA comprising an inverted repeat of a BS7 gene promoter. Saidbreeding pair further includes a second transgenic plant, which ishomozygous recessive for ms45, wherein the third exogenous nucleic acidmolecule comprises a nucleotide sequence encoding MS45 operably linkedto the BS7 gene promoter, and the fourth exogenous nucleic acid moleculecomprises a second hpRNA comprising an inverted repeat of the 5126 genepromoter. Upon crossing such first and second transgenic plants, malesterile progeny plants are obtained.

The present invention also relates to methods of producing a sterileplant. Such a method can be performed by crossing a breeding pair ofplants as disclosed herein. In one embodiment, the first plant of thebreeding pair contains a mutation inactivating a first endogenous geneof a pathway involved in male fertility, and the second plant contains asecond endogenous gene of the same or a different but convergent pathwayalso involved in the male sterility, wherein the progeny plants aredouble mutants and have a male sterile phenotype. In another embodiment,the method is performed using first and second transgenic plants, eachcontaining a transgene encoding an hpRNA that inactivates the respectiveendogenous fertility gene in the second and first transgenic plants,wherein progeny plants produced by crossing the parental plants exhibitthe sterile phenotype.

The present invention also relates to a method of producing a transgenicnon-human organism that exhibits a recessive phenotype, by breedingparental transgenic organisms that do not exhibit the recessivephenotype. For example, the invention provides methods of producing asterile progeny plant by crossing first and second transgenic plants,each of which is homozygous recessive for the same fertility gene,wherein, in the first transgenic plant, a fertility restorer gene isexpressed from a first promoter and an hpRNA is expressed thatsuppresses expression from a second promoter, and in the secondtransgenic plant, the fertility restorer gene is expressed from thesecond promoter, and a second hpRNA is expressed that suppressesexpression of the first promoter. The sterile progeny plants can befemale sterile or male sterile plants. For example, in a cross of afirst transgenic plant containing a first exogenous nucleic acidmolecule comprising a nucleotide sequence encoding MS45 operably linkedto a 5126 gene promoter, and a second exogenous nucleic acid moleculecomprising a first hpRNA including a nucleotide sequence of a BS7 genepromoter; and a second transgenic plant containing a third exogenousnucleic acid molecule comprising a nucleotide sequence encoding MS45operably linked to the BS7 gene promoter, and a fourth exogenous nucleicacid molecule comprising a second hpRNA including a nucleotide sequenceof the 5126 gene promoter, male sterile progeny are produced.Accordingly, the invention provides a plant produced by a method asdisclosed herein, for example, a male sterile plant.

The present invention further relates to a method of producing hybridplant seed. Such a method can be performed, for example, by pollinating(e.g., naturally, mechanically, or by hand) a male sterile plantproduced as disclosed herein with pollen of a male fertile plant thatcontains at least one dominant allele corresponding to the homozygousrecessive sterile genotype of the male sterile plant, whereby pollinatedmale sterile plants produce hybrid seed. As such, the invention alsoprovides hybrid seed produced by such a method. The present inventionrelates to a method of obtaining a hybrid plant by growing such hybridseed and, further, provides hybrid plants produced by growing suchhybrid seed.

The present invention further relates to a method of identifying afunction of a gene expressed in a cell. The gene expressed in the cellcan be any gene containing a promoter, including an endogenous gene,which contains an endogenous promoter. A method of identifying a genefunction can be performed, for example, by introducing into a cell inwhich the gene is expressed, a first exogenous nucleic acid moleculecomprising a nucleotide sequence encoding a hpRNA operably linked to afirst heterologous promoter, wherein the hpRNA comprises a nucleotidesequence of an endogenous promoter of the gene whose function is beingexamined, and wherein, upon expression, the hpRNA suppresses expressionof the gene; and detecting a change in a phenotype of the cell uponexpression of the hpRNA as compared to a wild type phenotype in theabsence of expression of the hpRNA, whereby the change in phenotypeidentifies the function of the gene. In one aspect, the method furtherincludes introducing into the cell a second exogenous nucleic acidmolecule comprising a nucleotide sequence encoding a polypeptide encodedby the gene operably linked to a second heterologous promoter, wherein,upon expression of the polypeptide encoded by the gene from the secondheterologous promoter, the wild type phenotype is restored.

A method of the invention can be practiced using single cells containingthe gene of interest, or can be practiced using an organism containingthe cell. The organism can be any organism of interest in which the geneof interest is expressed. In one embodiment, the cell is a plant cell,which can be a plant cell in vitro or can be one or more cells of aplant in situ. In one embodiment, the organism is a transgenic plant,which contains the first exogenous nucleic acid molecule stablyintegrated in its genome. In an aspect of this embodiment, thetransgenic plant further contains, integrated in its genome, a secondexogenous nucleic acid molecule (comprising a nucleotide sequenceencoding a polypeptide encoded by the gene of interest) operably linkedto a second heterologous promoter, wherein, upon expression of thesecond exogenous nucleic acid molecule from the second heterologouspromoter, the wild type phenotype is restored.

In some embodiments, the present invention addresses the difficulty inpropagating a plant having a homozygous recessive reproductive traitwithout losing the homozygous recessive condition in the resultingprogeny. This may be accomplished by introducing into a plant at leastone restoring transgene construct, operably linking (1) a firstnucleotide sequence comprising a functional copy of a gene thatcomplements the mutant phenotypic trait produced by the homozygousrecessive condition with (2) a second functional nucleotide sequencewhich interferes with the formation, function, or dispersal of the malegametes of the plant and is operably linked to amale-gamete-tissue-preferred promoter. This construct is maintained inthe hemizygous state and a plant containing such a construct is referredto herein as a maintainer. When the maintainer plant containing such alinked construct is used as a pollen donor to fertilize the homozygousrecessive plant, the only viable male gametes provided to the homozygousrecessive plant are those which contain the recessive allele, and do notcontain any component of the transgene construct. None of the pollengrains which contain the restoring transgene construct are viable, dueto the action of the linked second gene that prevents the formation ofviable pollen. Therefore, the progeny resulting from such a sexual crossare non transgenic with respect to this transgene construct.

While no viable pollen produced by the maintainer contains the restoringtransgene construct, 50% of the ovules (the female gamete) of themaintainer will contain the restoring transgene construct. Therefore,the maintainer can be propagated by self-fertilization, with therestoring transgene construct segregating such that it will be containedin 50% of the seed of the ear of a self fertilized maintainer. Bylinking the restoring transgene construct with a selectable marker, the50% of the seed containing the transgene can be isolated to propagatethe maintainer population, which remains homozygous for the recessivegene and hemizygous for the restoring transgene construct.

In a further embodiment, if the female gamete is prohibited from beingformed or functional, it will be desirable to link the gene capable ofcomplementing this mutant phenotype with an inducible promoter to aid inmaintenance of the maintainer plant. Such a plant, when exposed to theinducing condition, will have female fertility restored, and the plantmay then be self fertilized to produce progeny having the both thedesired recessive mutant trait and the restoring transgene construct.

While the invention is exemplified in plants, a person of skill in theart would recognize its applicability to other non-human organisms,including mammals. For example, the invention encompasses a method ofsuppressing a phenotype in progeny of a parental pair of non-humanorganisms, wherein (a) said phenotype is expressed in each of saidparents; (b) the genome of each parent is manipulated so as toinactivate a gene affecting the phenotype of interest; and (c) the geneinactivated in the first parent encodes a different gene product thanthe gene inactivated in the second parent.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the invention reflect the determination that the genotypeof an organism can be modified to contain dominant suppressor alleles ortransgene constructs that suppress (i.e., reduce, but not ablate) theactivity of a gene, wherein the phenotype of the organism is notsubstantially affected.

In some embodiments, the present invention is exemplified with respectto plant fertility, and more particularly with respect to plant malefertility. For example, plants may be genetically modified to contain atransgene construct encoding hairpin RNA (hpRNA) molecules that suppressthe expression of an endogenous male fertility gene without renderingthe plant male sterile.

In one example, Gene A and Gene B modulate sequential (though notnecessarily consecutive) steps in a pathway leading to a product. In afirst plant, Gene A is suppressed so as to reduce, but not ablate, GeneA activity. The pathway is not substantially inhibited, and thus thephenotype of said first plant is not affected. In a second plant, Gene Bis suppressed so as to reduce, but not ablate, Gene B activity. Thepathway is not substantially inhibited, and thus the phenotype of saidsecond plant is not affected. In progeny of a cross of said first andsecond plants, the combination of suppression of Gene A and Gene B leadsto loss of the product of the pathway and a change in phenotype.Suppression of Gene A and/or Gene B could be accomplished by use ofhairpin constructs (hpRNA) as described elsewhere herein.

In another example, Gene A and Gene B modulate steps of convergentpathways prior to the point of convergence, and the converged pathwayleads to a product. In a first plant, Gene A is suppressed so as toreduce, but not ablate, Gene A activity, and the phenotype of said firstplant is not affected. In a second plant, Gene B is suppressed so as toreduce, but not ablate, Gene B activity, and the phenotype of saidsecond plant is not affected. In progeny of a cross of said first andsecond plants, the combination of suppression of Gene A and Gene B leadsto loss of the product of the convergent pathways. Suppression of Gene Aand/or Gene B could be accomplished by use of hairpin constructs (hpRNA)as described elsewhere herein.

In certain embodiments, Gene A and Gene B modulate steps of pathwaysinvolved in plant fertility. In this way, for example, crosses ofphenotypically fertile plants expressing targeted hpRNA molecules cangenerate male sterile plants. For example, parental plants having ahomozygous recessive male sterile genotype can be transformed such thateach expresses a restorer male fertility gene from differentheterologous promoters and hpRNAs that suppress expression of therestorer gene in the other parental plant. Such parental plants, whichare fertile, can be crossed with each other to generate male sterileplants. This is exemplified by a pair of male-fertile plants, A and B.Each has a homozygous recessive male sterile genotype, ms45 ms45. PlantA is transformed with, in single or multiple constructs, a 5126 promoteroperably linked to a restorer MS45 gene, and an hpRNA specific for theBS7 promoter. Plant B is transformed with, in single or multipleconstructs, a BS7 promoter operably linked to a restorer MS45 gene, andan hpRNA specific for the 5126 promoter. Plant A and Plant B are eachmale-fertile due to the presence of the MS45 restorer. In a cross ofPlant A and Plant B, restoration of fertility is reversed due to theaction of the complementing hairpin constructs targeted to therespective promoters driving the restorer gene, and the progeny of saidcross are male-sterile. Such progeny are useful as females in hybridproduction. Wild-type pollen can restore fertility in the hybrid due tothe recessive nature of the ms45 allele.

Certain embodiments of the invention comprise a transgenic non-humanorganism having a homozygous recessive genotype that results in absenceof a particular phenotype of interest, said organism further comprising(a) a first exogenous nucleic acid molecule comprising a restorer genefor the particular phenotype, operably linked to a first promoter; and(b) a second exogenous nucleic acid molecule comprising a secondpromoter operably linked to a nucleotide sequence encoding a firsthairpin ribonucleic acid molecule (hpRNA), wherein the first hpRNAcomprises a nucleotide sequence of the first promoter or a nucleotidesequence of a third promoter, wherein said transgenic non-human organismexhibits the phenotype of interest.

The agriculture industry produces crops that are used to feed humans andanimals, and that are further used in other industries to prepareproducts as diverse as adhesives and explosives. Maize (corn), forexample, is used as human food, livestock feed (e.g., beef cattle, dairycattle, hogs, and poultry feed), and a raw material in industry. Fooduses of maize include consumption of maize kernels as well as productsof dry-milling and wet-milling industries (e.g., grits, meal, flour,maize starch, maize syrups, and dextrose). Maize oil is recovered frommaize germ, which is a by-product of the dry-milling and wet-millingindustries. Industrial uses of maize include production of ethanol,maize starch in the wet-milling industry and maize flour in thedry-milling industry. The industrial applications of maize starch andflour are based on their functional properties, including, for example,viscosity, film formation, adhesive properties, and ability to suspendparticles. Maize starch and flour have application in the paper andtextile industries, and also are used in adhesives, building materials,foundry binders, laundry starches, explosives, oil-well muds, and othermining applications.

Many crop plants, including rice, wheat, maize, tomatoes, and melons aregrown as hybrids, which exhibit greater vigor and improved qualities ascompared to the parental plants. The development of hybrids in a plantbreeding program requires, in general, the development of homozygousinbred lines, the crossing of these lines, and the evaluation of thecrosses. Pedigree breeding and recurrent selection breeding methods areused to develop inbred lines from breeding populations. For example,maize plant breeding programs combine the genetic backgrounds from twoor more inbred lines (or various other germplasm sources) into breedingpools, from which new inbred lines are developed by self-pollinating(selfing) and selection of desired phenotypes. The selected inbreds thenare crossed with other inbred lines and the hybrids from these crossesare evaluated to determine which of those have commercial potential. Assuch, plant breeding and hybrid development are expensive andtime-consuming processes.

Pedigree breeding starts with the crossing of two genotypes, each ofwhich may have one or more desirable characteristics that is lacking inthe other or which complements the other. If the two original parents donot provide all the desired characteristics, other sources can beincluded in the breeding population. Using this method, superior plantsare selected and selfed in successive generations until homogeneousplant lines are obtained. Recurrent selection breeding such asbackcrossing can be used to improve an inbred line and a hybrid can bemade using the inbreds. Backcrossing can be used to transfer a specificdesirable trait from one inbred or source to a second inbred that lacksthat trait, for example, by first crossing a superior inbred (recurrentparent) to a donor inbred (non-recurrent parent) that carries theappropriate gene (or genes) for the trait in question, crossing theprogeny of the first cross back to the superior recurrent parent, andselecting in the resultant progeny for the desired trait to betransferred from the non-recurrent parent. After five or more backcrossgenerations with selection for the desired trait, the progeny arehomozygous for loci controlling the characteristic being transferred,and are like the superior parent for essentially all other genes. Thelast backcross generation is selfed to give pure breeding progeny forthe gene being transferred.

A single cross hybrid (F1) results from the cross of two inbred lines(P1 and P2), each of which has a genotype that complements the genotypeof the other. In the development of commercial hybrids in a maize plantbreeding program, for example, only F1 hybrid plants are sought, as theyare more vigorous than their inbred parents. This hybrid vigor(heterosis) can be manifested in many polygenic traits such as increasedvegetative growth and increased yield. The development of a hybrid in amaize plant breeding program, for example, involves the selection ofplants from various germplasm pools for initial breeding crosses; theselfing of the selected plants from the breeding crosses for severalgenerations to produce a series of inbred lines, which, althoughdifferent from each other, breed true and are highly uniform; andcrossing the selected inbred lines with different inbred lines toproduce the hybrid F1 progeny. During the inbreeding process in maize,the vigor of the lines decreases, but is restored when two differentinbred lines are crossed to produce the hybrid plants. An importantconsequence of the homozygosity and homogeneity of the inbred lines isthat the F1 hybrid between a defined pair of inbred parental plantsalways is the same. As such, once the inbreds that provide a superiorhybrid are identified, the hybrid seed can be reproduced indefinitely aslong as the inbred parents are maintained.

Hybrid seed production requires elimination or inactivation of pollenproduced by the female parent. Incomplete removal or inactivation of thepollen provides the potential for selfing, raising the risk thatinadvertently self-pollinated seed will unintentionally be harvested andpackaged with hybrid seed. Once the seed is planted, the selfed plantscan be identified and selected; the selfed plants are geneticallyequivalent to the female inbred line used to produce the hybrid.Typically, the selfed plants are identified and selected based on theirdecreased vigor. For example, female selfed plants of maize areidentified by their less vigorous appearance for vegetative and/orreproductive characteristics, including shorter plant height, small earsize, ear and kernel shape, cob color, or other characteristics. Selfedlines also can be identified using molecular marker analyses (see, e.g.,Smith and Wych, (1995) Seed Sci. Technol. 14:1-8). Using such methods,the homozygosity of the self-pollinated line can be verified byanalyzing allelic composition at various loci in the genome.

Because hybrid plants are important and valuable field crops, plantbreeders are continually working to develop high-yielding hybrids thatare agronomically sound based on stable inbred lines. The availabilityof such hybrids allows a maximum amount of crop to be produced with theinputs used, while minimizing susceptibility to pests and environmentalstresses. To accomplish this goal, the plant breeder must developsuperior inbred parental lines for producing hybrids by identifying andselecting genetically unique individuals that occur in a segregatingpopulation. The present invention contributes to this goal, for exampleby providing plants that, when crossed, generate male sterile progeny,which can be used as female parental plants for generating hybridplants.

A large number of genes have been identified as being tassel preferredin their expression pattern using traditional methods and more recenthigh-throughput methods. The correlation of function of these genes withimportant biochemical or developmental processes that ultimately lead tofertile pollen is arduous when approaches are limited to classicalforward or reverse genetic mutational analysis. As disclosed herein,suppression approaches in maize provide an alternative rapid means toidentify genes that are directly related to pollen development in maize.The well-characterized maize male fertility gene, MS45, and severalanther-preferred genes of unknown function were used to evaluate theefficacy of generating male sterility using post-transcriptional genesilencing (PTGS; see, for example, Kooter, et al., (1999) Trends PlantSci. 4:340-346) or transcriptional gene silencing (TGS; see, forexample, Mette, et al., (2000) EMBO J 19:5194-5201) approaches.

To examine PTGS, hairpin-containing RNAi constructs that have stemstructures composed of inverted repeats of the anther-expressed cDNAsequences, and a loop containing either a non-homologous coding sequenceor a splicable intron from maize, were introduced into maize.

To examine TGS as an approach to knock out anther gene function, asecond set of constructs was generated in which the promoters of theanther-specific gene sequences formed the stem and a non-homologoussequence formed the loop. The constructs were expressed usingconstitutive promoters and anther-preferred promoters.

Contrasting fertility phenotypes were observed, depending on the type ofhairpin construct expressed. Plants expressing the PTGS constructs weremale fertile. In contrast, plants expressing the TGS constructs weremale sterile, and lacked MS45 mRNA and protein. Further, the sterilityphenotype of the plants containing the hpRNA specific for the MS45promoter (i.e., the TGS constructs) was reversed when MS45 was expressedfrom heterologous promoters in these plants. These results demonstratethat TGS provides a tool for rapidly correlating gene expression withfunction of unknown genes such as anther-expressed monocot genes.

Accordingly, the invention provides breeding pairs of plants, whereinthe plants comprising the breeding pair are fertile (i.e., male fertileand female fertile), and wherein progeny produced by crossing thebreeding pair of plants are sterile (e.g., male sterile). As disclosedherein, a breeding pair of plants of the invention can include, forexample, a first plant having an inactivated first endogenous fertilitygene, wherein the first plant is fertile; and a second plant having aninactivated second endogenous fertility gene, wherein the second plantis fertile. Such a breeding pair is characterized, in part, in that ifthe first endogenous fertility gene is a male fertility gene, then thesecond endogenous fertility gene also is a male fertility gene; whereasif the first endogenous fertility gene is a female fertility gene, thenthe second endogenous fertility gene also is a female fertility gene.

As used herein, the term “endogenous”, when used in reference to a gene,means a gene that is normally present in the genome of cells of aspecified organism, and is present in its normal state in the cells(i.e., present in the genome in the state in which it normally ispresent in nature). The term “exogenous” is used herein to refer to anymaterial that is introduced into a cell. The term “exogenous nucleicacid molecule” or “transgene” refers to any nucleic acid molecule thateither is not normally present in a cell genome or is introduced into acell. Such exogenous nucleic acid molecules generally are recombinantnucleic acid molecules, which are generated using recombinant DNAmethods as disclosed herein or otherwise known in the art. In variousembodiments, a transgenic non-human organism as disclosed herein, cancontain, for example, a first transgene and a second transgene. Suchfirst and second transgenes can be introduced into a cell, for example,a progenitor cell of a transgenic organism, either as individual nucleicacid molecules or as a single unit (e.g., contained in different vectorsor contained in a single vector, respectively). In either case,confirmation may be made that a cell from which the transgenic organismis to be derived contains both of the transgenes using routine andwell-known methods such as expression of marker genes or nucleic acidhybridization or PCR analysis. Alternatively, or additionally,confirmation of the presence of transgenes may occur later, for example,after regeneration of a plant from a putatively transformed cell.

An endogenous fertility gene of a plant of a breeding pair of theinvention can be inactivated due, for example, (1) to a mutation of theendogenous gene such that the function of a product encoded by the geneis suppressed (e.g., the gene product is not expressed or is expressedat a level that is insufficient to mediate its full effect in the plantor plant cell); or (2) to expression of an exogenous nucleic acidmolecule that reduces or inhibits expression of the gene product encodedby the endogenous gene. As such, the term “inactivated” is used broadlyherein to refer to any manipulation of an endogenous gene, or a cellcontaining the gene, such that the function mediated by a productencoded by the gene is suppressed. It should further be recognized that,regardless of whether the inactivated endogenous gene has reducedactivity or is completely inactive, the desired relevant phenotype ismaintained. As such, reference to an inactivated male fertility gene ina parental plant defined as having a male fertile phenotype can include,for example, a male fertility gene that is expressed at a level that islower than normal, but sufficient to maintain fertility of the parentalplant, or a male fertility gene that is completely inactive, and whereinfertility of the parental plant is maintained due to expression of asecond gene product.

Mutation of an endogenous gene that results in suppression of the genefunction can be effected, for example, by deleting or inserting one or afew nucleotides into the nucleotide sequence of the gene (e.g., into thepromoter, coding sequence, or intron), by substituting one or a fewnucleotides in the gene with other different nucleotides, or by knockingout the gene (e.g., by homologous recombination using an appropriatetargeting vector). Plants having such mutations in both alleles can beobtained, for example, using crossing methods as disclosed herein orotherwise known in the art. Inactivation of an endogenous gene thatresults in suppression of the gene function also can be effected byintroduction into cells of the plant of a transgene that suppressesexpression of the endogenous gene or a product expressed from theendogenous gene (e.g., an encoded polypeptide), or a transgene thatencodes a product (e.g., an RNA) that suppresses expression of theendogenous gene or a product encoded by the endogenous gene in cells ofthe plant in which the gene normally is expressed.

By way of example, inactivation of endogenous fertility genes can beeffected by expressing hairpin RNA molecules (hpRNA) in cells of thereproductive organs of a plant (e.g., stamen cells where the endogenousfertility genes to be inactivated are male fertility genes). The stamen,which comprises the male reproductive organ of plants, includes variouscell types, including, for example, the filament, anther, tapetum, andpollen. The hpRNAs useful for purposes of the present invention aredesigned to include inverted repeats of a promoter of the endogenousgene to be inactivated; hpRNAs having the ability to suppress expressionof a gene have been described (see, e.g., Matzke, et al., (2001) Curr.Opin. Genet. Devel. 11:221-227; Scheid, et al., (2002) Proc. Natl. Acad.Sci., USA 99:13659-13662; Waterhouse and Helliwell (2003) Nature ReviewsGenetics 4:29-38; Aufsaftz, et al., (2002) Proc. Nat'l. Acad. Sci.99(4):16499-16506; Sijen, et al., (2001) Curr. Biol. 11:436-440). Asdisclosed herein, the use of stamen-specific or stamen-preferredpromoters, including anther-specific promoters, pollen-specificpromoters, tapetum-specific promoters, and the like, allows forexpression of hpRNAs in plants (particularly in male reproductive cellsof the plant), wherein the hpRNA suppresses expression of an endogenousfertility gene, thereby inactivating expression of the endogenousfertility gene. As such, suppression using an hpRNA specific for apromoter that directs expression of a fertility gene provides a means toinactivate an endogenous fertility gene.

In one embodiment, a breeding pair of plants of the invention caninclude a first plant, which contains a first exogenous nucleic acidmolecule comprising a promoter operably linked to a nucleotide sequenceencoding a first hpRNA, wherein the first hpRNA comprises a nucleotidesequence comprising an inverted repeat of the first endogenous fertilitygene promoter, and wherein, upon expression, the first hpRNA suppressesexpression of the first endogenous fertility gene; and a second plant,which contains a second exogenous nucleic acid molecule comprising apromoter operably linked to a nucleotide sequence encoding a secondhpRNA, wherein the second hpRNA comprises a nucleotide sequencecomprising an inverted repeat of the second endogenous fertility genepromoter, and wherein, upon expression, the second hpRNA suppressesexpression of the second endogenous fertility gene. According to thepresent invention, the first and/or second exogenous nucleic acid can,but need not, be stably integrated in the genome of cells of the firstand/or second plant, respectively. Such first and second plants of thebreeding pair are characterized, in part, in that each is fertile, andis further characterized in that, when crossed, the progeny of suchcross is sterile (e.g., male sterile).

The terms “first”, “second”, “third”, and “fourth” are used herein onlyto clarify relationships of various cells and molecules or todistinguish different types of a molecule, and, unless specificallyindicated otherwise, are not intended to indicate any particular order,importance, or quantitative feature. For example, and unlessspecifically indicated otherwise, reference to a “first” plantcontaining a “first endogenous gene” is intended to indicate only thatthe specified gene is present in the specified plant. By way of a secondexample, and unless specifically indicated otherwise, reference to a“first plant containing a first transgene and a second transgene” isintended to indicate only that said plant contains two exogenous nucleicacid molecules that are different from each other.

As used herein, the term “nucleic acid molecule” or “polynucleotide” or“nucleotide sequence” refers broadly to a sequence of two or moredeoxyribonucleotides or ribonucleotides that are linked together by aphosphodiester bond. As such, the terms include RNA and DNA, which canbe a gene or a portion thereof, a cDNA, a synthetic polydeoxyribonucleicacid sequence, or the like, and can be single-stranded ordouble-stranded, as well as a DNA/RNA hybrid. Furthermore, the terms areused herein to include naturally-occurring nucleic acid molecules, whichcan be isolated from a cell, as well as synthetic molecules, which canbe prepared, for example, by methods of chemical synthesis or byenzymatic methods such as by the polymerase chain reaction (PCR). Theterm “recombinant” is used herein to refer to a nucleic acid moleculethat is manipulated outside of a cell, including two or more linkedheterologous nucleotide sequences. The term “heterologous” is usedherein to refer to nucleotide sequence that are not normally linked innature or, if linked, are linked in a different manner than thatdisclosed. For example, reference to a transgene comprising a codingsequence operably linked to a heterologous promoter means that thepromoter is one that does not normally direct expression of thenucleotide sequence in a specified cell in nature.

In general, the nucleotides comprising an exogenous nucleic acidmolecule (transgene) are naturally occurring deoxyribonucleotides, suchas adenine, cytosine, guanine or thymine linked to 2′-deoxyribose, orribonucleotides such as adenine, cytosine, guanine or uracil linked toribose. However, a nucleic acid molecule or nucleotide sequence also cancontain nucleotide analogs, including non-naturally-occurring syntheticnucleotides or modified naturally-occurring nucleotides. Such nucleotideanalogs are well known in the art and commercially available, as arepolynucleotides containing such nucleotide analogs (Lin, et al., (1994)Nucl. Acids Res. 22:5220-5234; Jellinek, et al., (1995) Biochemistry34:11363-11372; Pagratis, et al., (1997) Nature Biotechnol. 15:68-73).Similarly, the covalent bond linking the nucleotides of a nucleotidesequence generally is a phosphodiester bond, but also can be, forexample, a thiodiester bond, a phosphorothioate bond, a peptide-likebond or any other bond known to those in the art as useful for linkingnucleotides to produce synthetic polynucleotides (see, for example, Tam,et al., (1994) Nucl. Acids Res. 22:977-986; Ecker and Crooke, (1995)BioTechnology 13:351360). The incorporation of non-naturally occurringnucleotide analogs or bonds linking the nucleotides or analogs can beparticularly useful where the nucleic acid molecule is to be exposed toan environment that can contain a nucleolytic activity, including, forexample, a plant tissue culture medium or in a plant cell, since themodified molecules can be less susceptible to degradation.

A nucleotide sequence containing naturally-occurring nucleotides andphosphodiester bonds can be chemically synthesized or can be producedusing recombinant DNA methods, using an appropriate polynucleotide as atemplate. In comparison, a nucleotide sequence containing nucleotideanalogs or covalent bonds other than phosphodiester bonds generally ischemically synthesized, although an enzyme such as T7 polymerase canincorporate certain types of nucleotide analogs into a polynucleotideand, therefore, can be used to produce such a polynucleotiderecombinantly from an appropriate template (Jellinek, et al., supra,1995).

An exogenous nucleic acid molecule can comprise operably linkednucleotide sequences such as a promoter operably linked to a nucleotidesequence encoding an hpRNA, or a promoter linked to a nucleotidesequence encoding a male fertility gene product. The term “operablylinked” is used herein to refer to two or more molecules that, whenjoined together, generate a molecule that shares features characteristicof each of the individual molecules. For example, when used in referenceto a promoter (or other regulatory element) and a second nucleotidesequence encoding a gene product, the term “operably linked” means thatthe regulatory element is positioned with respect to the secondnucleotide sequence such that transcription or translation of theisolated nucleotide sequence is under the influence of the regulatoryelement. When used in reference to a fusion protein comprising a firstpolypeptide and one or more additional polypeptides, the term “operablylinked” means that each polypeptide component of the fusion (chimeric)protein exhibits some or all of a function that is characteristic of thepolypeptide component (e.g., a cell compartment localization domain anda enzymatic activity). In another example, two operably linkednucleotide sequences, each of which encodes a polypeptide, can be suchthat the coding sequences are in frame and, therefore, upontranscription and translation, result in production of two polypeptides,which can be two separate polypeptides or a fusion protein.

Where an exogenous nucleic acid molecule includes a promoter operablylinked to a nucleotide sequence encoding an RNA or polypeptide ofinterest, the exogenous nucleic acid molecule can be referred to as anexpressible exogenous nucleic acid molecule (or transgene). The term“expressible” is used herein because, while such a nucleotide sequencecan be expressed from the promoter, it need not necessarily actually beexpressed at a particular point in time. For example, where a promoterof an expressible transgene is an inducible promoter lacking basalactivity, an operably linked nucleotide sequence encoding an RNA orpolypeptide of interest is expressed only following exposure to anappropriate inducing agent.

Transcriptional promoters generally act in a position- andorientation-dependent manner, and usually are positioned at or withinabout five nucleotides to about fifty nucleotides 5′ (upstream) of thestart site of transcription of a gene in nature. In comparison,enhancers can act in a relatively position- or orientation-independentmanner, and can be positioned several hundred or thousand nucleotidesupstream or downstream from a transcription start site, or in an intronwithin the coding region of a gene, yet still be operably linked to thecoding region so as to enhance transcription. The relative positions andorientations of various regulatory elements in addition to a promoter,including the positioning of a transcribed regulatory sequence such asan internal ribosome entry site, or a translated regulatory element suchas a cell compartmentalization domain in an appropriate reading frame,are well known, and methods for operably linking such elements areroutine in the art (see, for example, Sambrook, et al., “MolecularCloning: A laboratory manual” (Cold Spring Harbor Laboratory Press1989); Ausubel, et al., “Current Protocols in Molecular Biology” (JohnWiley and Sons, Baltimore Md. 1987, and supplements through 1995)).

Promoters useful for expressing a nucleic acid molecule of interest canbe any of a range of naturally-occurring promoters known to be operativein plants or animals, as desired. Promoters that direct expression incells of male or female reproductive organs of a plant are useful forgenerating a transgenic plant or breeding pair of plants of theinvention. The promoters useful in the present invention can includeconstitutive promoters, which generally are active in most or alltissues of a plant; inducible promoters, which generally are inactive orexhibit a low basal level of expression, and can be induced to arelatively high activity upon contact of cells with an appropriateinducing agent; tissue-specific (or tissue-preferred) promoters, whichgenerally are expressed in only one or a few particular cell types(e.g., plant anther cells); and developmental- or stage-specificpromoters, which are active only during a defined period during thegrowth or development of a plant. Often promoters can be modified, ifnecessary, to vary the expression level. Certain embodiments comprisepromoters exogenous to the species being manipulated. For example, theMs45 gene introduced into ms45 ms45 maize germplasm may be driven by apromoter isolated from another plant species; a hairpin construct maythen be designed to target the exogenous plant promoter, reducing thepossibility of hairpin interaction with non-target, endogenous maizepromoters.

Exemplary constitutive promoters include the 35S cauliflower mosaicvirus (CaMV) promoter promoter (Odell, et al., (1985) Nature313:810-812), the maize ubiquitin promoter (Christensen, et al., (1989)Plant Mol. Biol. 12:619-632 and Christensen, et al., (1992) Plant Mol.Biol. 18:675-689); the core promoter of the Rsyn7 promoter and otherconstitutive promoters disclosed in WO 99/43838 and U.S. Pat. No.6,072,050; rice actin (McElroy, et al., (1990) Plant Cell 2:163-171);pEMU (Last, et al., (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten,et al., (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No.5,659,026); rice actin promoter (U.S. Pat. No. 5,641,876; WO 00/70067),maize histone promoter (Brignon, et al., (1993) Plant Mol Bio22(6):1007-1015; Rasco-Gaunt, et al., (2003) Plant Cell Rep. 21(6):569-576) and the like. Other constitutive promoters include, forexample, those described in U.S. Pat. Nos. 5,608,144 and 6,177,611, andPCT publication WO 03/102198.

Tissue-specific, tissue-preferred, or stage-specific regulatory elementsfurther include, for example, the AGL8/FRUITFULL regulatory element,which is activated upon floral induction (Hempel, et al., (1997)Development 124:3845-3853); root-specific regulatory elements such asthe regulatory elements from the RCP1 gene and the LRP1 gene (Tsugekiand Fedoroff, (1999) Proc. Natl. Acad., USA 96:12941-12946; Smith andFedoroff, (1995) Plant Cell 7:735-745); flower-specific regulatoryelements such as the regulatory elements from the LEAFY gene and theAPETALA1 gene (Blazquez, et al., (1997) Development 124:3835-3844;Hempel, et al., supra, 1997); seed-specific regulatory elements such asthe regulatory element from the oleosin gene (Plant, et al., (1994)Plant Mol. Biol. 25:193-205), and dehiscence zone specific regulatoryelement. Additional tissue-specific or stage-specific regulatoryelements include the Zn13 promoter, which is a pollen-specific promoter(Hamilton, et al., (1992) Plant Mol. Biol. 18:211-218); the UNUSUALFLORAL ORGANS (UFO) promoter, which is active in apical shoot meristem;the promoter active in shoot meristems (Atanassova, et al., (1992) PlantJ. 2:291), the cdc2 promoter and cyc07 promoter (see, for example, Ito,et al., (1994) Plant Mol. Biol. 24:863-878; Martinez, et al., (1992)Proc. Natl. Acad. Sci., USA 89:7360); the meristematic-preferred meri-5and H3 promoters (Medford, et al., (1991) Plant Cell 3:359; Terada, etal., (1993) Plant J. 3:241); meristematic and phloem-preferred promotersof Myb-related genes in barley (Wissenbach, et al., (1993) Plant J.4:411); Arabidopsis cyc3aAt and cyc1At (Shaul, et al., (1996) Proc.Natl. Acad. Sci. 93:4868-4872); C. roseus cyclins CYS and CYM (Ito, etal., (1997) Plant J. 11:983-992); and Nicotiana CyclinB1 (Trehin, etal., (1997) Plant Mol. Biol. 35:667-672); the promoter of the APETALA3gene, which is active in floral meristems (Jack, et al., (1994) Cell76:703; Hempel, et al., supra, 1997); a promoter of an agamous-like(AGL) family member, for example, AGL8, which is active in shootmeristem upon the transition to flowering (Hempel, et al., supra, 1997);floral abscission zone promoters; L1-specific promoters; theripening-enhanced tomato polygalacturonase promoter (Nicholass, et al.,(1995) Plant Mol. Biol. 28:423-435), the E8 promoter (Deikman, et al.,(1992) Plant Physiol. 100:2013-2017), and the fruit-specific 2A1promoter, U2 and U5 snRNA promoters from maize, the Z4 promoter from agene encoding the Z4 22 kD zein protein, the Z10 promoter from a geneencoding a 10 kD zein protein, a Z27 promoter from a gene encoding a 27kD zein protein, the A20 promoter from the gene encoding a 19 kD zeinprotein, and the like. Additional tissue-specific promoters can beisolated using well known methods (see, e.g., U.S. Pat. No. 5,589,379).Shoot-preferred promoters include shoot meristem-preferred promoterssuch as promoters disclosed in Weigel, et al., (1992) Cell 69:843-859(Accession Number M91208); Accession Number AJ131822; Accession NumberZ71981; Accession Number AF049870 and shoot-preferred promotersdisclosed in McAvoy, et al., (2003) Acta Hort. (ISHS) 625:379-385.Inflorescence-preferred promoters include the promoter of chalconesynthase (Van der Meer, et al., (1992) Plant J. 2(4):525-535),anther-specific LAT52 (Twell, et al., (1989) Mol. Gen. Genet.217:240-245), pollen-specific Bp4 (Albani, et al., (1990) Plant MolBiol. 15:605, maize pollen-specific gene Zm13 (Hamilton, et al., (1992)Plant Mol. Biol. 18:211-218; Guerrero, et al., (1993) Mol. Gen. Genet.224:161-168), microspore-specific promoters such as the apg genepromoter (Twell, et al., (1993) Sex. Plant Reprod. 6:217-224), andtapetum-specific promoters such as the TA29 gene promoter (Mariani, etal., (1990) Nature 347:737; U.S. Pat. No. 6,372,967), and otherstamen-specific promoters such as the MS45 gene promoter, 5126 genepromoter, BS7 gene promoter, PG47 gene promoter (U.S. Pat. No.5,412,085; U.S. Pat. No. 5,545,546; Plant J 3(2):261-271 (1993)), SGB6gene promoter (U.S. Pat. No. 5,470,359), G9 gene promoter (U.S. Pat.Nos. 5,8937,850; 5,589,610), SB200 gene promoter (WO 02/26789), or thelike (see, Example 1). Tissue-preferred promoters of interest furtherinclude a sunflower pollen-expressed gene SF3 (Baltz, et al., (1992) ThePlant Journal 2:713-721), B. napus pollen specific genes (Arnoldo, etal., (1992) J. Cell. Biochem, Abstract Number Y101204). Tissue-preferredpromoters further include those reported by Yamamoto, et al., (1997)Plant J. 12(2):255-265 (psaDb); Kawamata, et al., (1997) Plant CellPhysiol. 38(7):792-803 (PsPAL1); Hansen, et al., (1997) Mol. Gen Genet.254(3):337-343 (ORF13); Russell, et al., (1997) Transgenic Res.6(2):157-168 (waxy or ZmGBS; 27 kDa zein, ZmZ27; osAGP; osGT1);Rinehart, et al., (1996) Plant Physiol. 112(3):1331-1341 (Fbl2A fromcotton); Van Camp, et al., (1996) Plant Physiol. 112(2):525-535(Nicotiana SodA1 and SodA2); Canevascini, et al., (1996) Plant Physiol.112(2):513-524 (Nicotiana Itp1); Yamamoto, et al., (1994) Plant CellPhysiol. 35(5):773-778 (Pinus cab-6 promoter); Lam (1994) Results Probl.Cell Differ. 20:181-196; Orozco, et al., (1993) Plant Mol Biol.23(6):1129-1138 (spinach rubisco activase (Rca)); Matsuoka, et al.,(1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590 (PPDK promoter); andGuevara-Garcia, et al., (1993) Plant J. 4(3):495-505 (Agrobacterium pmaspromoter). A tissue-specific promoter that is active in cells of male orfemale reproductive organs can be particularly useful in certain aspectsof the present invention.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See, Thompson, et al., (1989)BioEssays 10:108. Such seed-preferred promoters include, but are notlimited to, Cim1 (cytokinin-induced message), cZ19B1 (maize 19 kDazein), mi1ps (myo-inositol-1-phosphate synthase); see, WO 00/11177 andU.S. Pat. No. 6,225,529. Gamma-zein is an endosperm-specific promoter.Globulin-1 (Glob-1) is a representative embryo-specific promoter. Fordicots, seed-specific promoters include, but are not limited to, beanβ-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and thelike. For monocots, seed-specific promoters include, but are not limitedto, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, gamma-zein, waxy,shrunken 1, shrunken 2, globulin 1, etc. See also, WO 00/12733 and U.S.Pat. No. 6,528,704, where seed-preferred promoters from end1 and end2genes are disclosed. Additional embryo specific promoters are disclosedin Sato, et al., (1996) Proc. Natl. Acad. Sci. 93:8117-8122 (ricehomeobox, OSH1); and Postma-Haarsma, et al., (1999) Plant Mol. Biol.39:257-71 (rice KNOX genes). Additional endosperm specific promoters aredisclosed in Albani, et al., (1984) EMBO 3:1405-15; Albani, et al.,(1999) Theor. Appl. Gen. 98:1253-62; Albani, et al., (1993) Plant J.4:343-55; Mena, et al., (1998) The Plant Journal 116:53-62 (barley DOF);Opsahl-Ferstad, et al., (1997) Plant J 12:235 -46 (maize Esr); and Wu,et al., (1998) Plant Cell Physiology 39:885-889 (rice GluA-3, GluB-1,NRP33, RAG-1).

An inducible regulatory element is one that is capable of directly orindirectly activating transcription of one or more DNA sequences orgenes in response to an inducer. The inducer can be a chemical agentsuch as a protein, metabolite, growth regulator, herbicide or phenoliccompound; or a physiological stress, such as that imposed directly byheat, cold, salt, or toxic elements, or indirectly through the action ofa pathogen or disease agent such as a virus; or other biological orphysical agent or environmental condition. A plant cell containing aninducible regulatory element may be exposed to an inducer by externallyapplying the inducer to the cell or plant such as by spraying, watering,heating or similar methods. An inducing agent useful for inducingexpression from an inducible promoter is selected based on theparticular inducible regulatory element. In response to exposure to aninducing agent, transcription from the inducible regulatory elementgenerally is initiated de novo or is increased above a basal orconstitutive level of expression. Typically the protein factor thatbinds specifically to an inducible regulatory element to activatetranscription is present in an inactive form which is then directly orindirectly converted to the active form by the inducer. Any induciblepromoter can be used in the instant invention (see, Ward, et al., (1993)Plant Mol. Biol. 22:361-366).

Examples of inducible regulatory elements include a metallothioneinregulatory element, a copper-inducible regulatory element, or atetracycline-inducible regulatory element, the transcription from whichcan be effected in response to divalent metal ions, copper ortetracycline, respectively (Furst, et al., (1988) Cell 55:705-717; Mett,et al., (1993) Proc. Natl. Acad. Sci., USA 90:4567-4571; Gatz, et al.,(1992) Plant J. 2:397-404; Roder, et al., (1994) Mol. Gen. Genet.243:32-38). Inducible regulatory elements also include an ecdysoneregulatory element or a glucocorticoid regulatory element, thetranscription from which can be effected in response to ecdysone orother steroid (Christopherson, et al., (1992) Proc. Natl. Acad. Sci.,USA 89:6314-6318; Schena, et al., (1991) Proc. Natl. Acad. Sci., USA88:10421-10425; U.S. Pat. No. 6,504,082); a cold responsive regulatoryelement or a heat shock regulatory element, the transcription of whichcan be effected in response to exposure to cold or heat, respectively(Takahashi, et al., (1992) Plant Physiol. 99:383-390); the promoter ofthe alcohol dehydrogenase gene (Gerlach, et al., (1982) PNAS USA79:2981-2985; Walker, et al., (1987) PNAS 84(19):6624-6628), inducibleby anaerobic conditions; and the light-inducible promoter derived fromthe pea rbcS gene or pea psaDb gene (Yamamoto, et al., (1997) Plant J.12(2):255-265); a light-inducible regulatory element (Feinbaum, et al.,(1991) Mol. Gen. Genet. 226:449; Lam and Chua, (1990) Science 248:471;Matsuoka, et al., (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590;Orozco, et al., (1993) Plant Mol. Bio. 23(6):1129-1138), a plant hormoneinducible regulatory element (Yamaguchi-Shinozaki, et al., (1990) PlantMol. Biol. 15:905; Kares, et al., (1990) Plant Mol. Biol. 15:225), andthe like. An inducible regulatory element also can be the promoter ofthe maize In2-1 or In2-2 gene, which responds to benzenesulfonamideherbicide safeners (Hershey, et al., (1991) Mol. Gen. Gene. 227:229-237;Gatz, et al., (1994) Mol. Gen. Genet. 243:32-38), and the Tet repressorof transposon Tn10 (Gatz, et al., (1991) Mol. Gen. Genet. 227:229-237).Stress inducible promoters include salt/water stress-inducible promoterssuch as P5CS (Zang, et al., (1997) Plant Sciences 129:81-89);cold-inducible promoters, such as, cor15a (Hajela, et al., (1990) PlantPhysiol. 93:1246-1252), cor15b (Wlihelm, et al., (1993) Plant Mol Biol23:1073-1077), wsc120 (Ouellet, et al., (1998) FEBS Lett. 423-324-328),ci7 (Kirch, et al., (1997) Plant Mol Biol. 33:897-909), ci21A(Schneider, et al., (1997) Plant Physiol. 113:335-45); drought-induciblepromoters, such as, Trg-31 (Chaudhary, et al., (1996) Plant Mol. Biol.30:1247-57), rd29 (Kasuga, et al., (1999) Nature Biotechnology18:287-291); osmotic inducible promoters, such as Rab17 (Vilardell, etal., (1991) Plant Mol. Biol. 17:985-93) and osmotin (Raghothama, et al.,(1993) Plant Mol Biol 23:1117-28); and heat inducible promoters, such asheat shock proteins (Barros, et al., (1992) Plant Mol. 19:665-75; Marrs,et al., (1993) Dev. Genet. 14:27-41), smHSP (Waters, et al., (1996) J.Experimental Botany 47:325-338), and the heat-shock inducible elementfrom the parsley ubiquitin promoter (WO 03/102198). Otherstress-inducible promoters include rip2 (U.S. Pat. No. 5,332,808 and USPublication Number 2003/0217393) and rd29a (Yamaguchi-Shinozaki, et al.,(1993) Mol. Gen. Genetics 236:331-340). Certain promoters are inducibleby wounding, including the Agrobacterium pmas promoter (Guevara-Garcia,et al., (1993) Plant J. 4(3):495-505) and the Agrobacterium ORF13promoter (Hansen, et al., (1997) Mol. Gen. Genet. 254(3):337-343).

Additional regulatory elements active in plant cells and useful in themethods or compositions of the invention include, for example, thespinach nitrite reductase gene regulatory element (Back, et al., (1991)Plant Mol. Biol. 17:9); a gamma zein promoter, an oleosin ole16promoter, a globulin I promoter, an actin I promoter, an actin clpromoter, a sucrose synthetase promoter, an INOPS promoter, an EXM5promoter, a globulin2 promoter, a b-32, ADPG-pyrophosphorylase promoter,an LtpI promoter, an Ltp2 promoter, an oleosin ole17 promoter, anoleosin ole18 promoter, an actin 2 promoter, a pollen-specific proteinpromoter, a pollen-specific pectate lyase gene promoter or PG47 genepromoter, an anther specific RTS2 gene promoter, SGB6 gene promoter, orG9 gene promoter, a tapetum specific RAB24 gene promoter, ananthranilate synthase alpha subunit promoter, an alpha zein promoter, ananthranilate synthase beta subunit promoter, a dihydrodipicolinatesynthase promoter, a Thi I promoter, an alcohol dehydrogenase promoter,a cab binding protein promoter, an H3C4 promoter, a RUBISCO SS starchbranching enzyme promoter, an actin3 promoter, an actin7 promoter, aregulatory protein GF14-12 promoter, a ribosomal protein L9 promoter, acellulose biosynthetic enzyme promoter, an S-adenosyl-L-homocysteinehydrolase promoter, a superoxide dismutase promoter, a C-kinase receptorpromoter, a phosphoglycerate mutase promoter, a root-specific RCc3 mRNApromoter, a glucose-6 phosphate isomerase promoter, apyrophosphate-fructose 6-phosphate-1-phosphotransferase promoter, abeta-ketoacyl-ACP synthase promoter, a 33 kDa photosystem 11 promoter,an oxygen evolving protein promoter, a 69 kDa vacuolar ATPase subunitpromoter, a glyceraldehyde-3-phosphate dehydrogenase promoter, an ABA-and ripening-inducible-like protein promoter, a phenylalanine ammonialyase promoter, an adenosine triphosphatase S-adenosyl-L-homocysteinehydrolase promoter, a chalcone synthase promoter, a zein promoter, aglobulin-1 promoter, an auxin-binding protein promoter, a UDP glucoseflavonoid glycosyl-transferase gene promoter, an NTI promoter, an actinpromoter, and an opaque 2 promoter.

An exogenous nucleic acid molecule can be introduced into a cell as anaked DNA molecule, can be incorporated in a matrix such as a liposomeor a particle such as a viral particle, or can be incorporated into avector. Incorporation of the polynucleotide into a vector can facilitatemanipulation of the polynucleotide, or introduction of thepolynucleotide into a plant cell. Accordingly, the vector can be derivedfrom a plasmid or can be a viral vector such as a T-DNA vector (Horsch,et al., (1985) Science 227:1229-1231). If desired, the vector caninclude components of a plant transposable element, for example, a Dstransposon (Bancroft and Dean, (1993) Genetics 134:1221-1229) or an Spmtransposon (Aarts, et al., (1995) Mol. Gen. Genet. 247:555-564). Inaddition to containing the transgene of interest, the vector also cancontain various nucleotide sequences that facilitate, for example,rescue of the vector from a transformed plant cell; passage of thevector in a host cell, which can be a plant, animal, bacterial, orinsect host cell; or expression of an encoding nucleotide sequence inthe vector, including all or a portion of a rescued coding region. Assuch, a vector can contain any of a number of additional transcriptionand translation elements, including constitutive and induciblepromoters, enhancers, and the like (see, for example, Bitter, et al.,(1987) Meth. Enzymol. 153:516-544). For example, a vector can containelements useful for passage, growth or expression in a bacterial system,including a bacterial origin of replication; a promoter, which can be aninducible promoter; and the like. A vector also can contain one or morerestriction endonuclease recognition and cleavage sites, including, forexample, a polylinker sequence, to facilitate insertion or removal of atransgene.

In addition to, or alternatively to, a nucleotide sequence relevant to afertility gene (e.g., an hpRNA comprising an inverted repeat of afertility gene promoter, or a coding sequence of a fertility gene, aloneor operably linked to a heterologous promoter), an exogenous nucleicacid molecule, or a vector containing such a transgene, can contain oneor more other expressible nucleotide sequences encoding an RNA or apolypeptide of interest. For example, the additional nucleotide sequencecan encode an antisense nucleic acid molecule; an enzyme such asβ-galactosidase, β-glucuronidase, luciferase, alkaline phosphatase,glutathione α-transferase, chloramphenicol acetyltransferase, guaninexanthine phosphoribosyltransferase, and neomycin phosphotransferase; aviral polypeptide or a peptide portion thereof; or a plant growth factoror hormone.

In certain embodiments, the expression vector contains a gene encoding aselection marker which is functionally linked to a promoter thatcontrols transcription initiation. For a general description of plantexpression vectors and reporter genes, see, Gruber, et al., “Vectors forPlant Transformation” in Methods of Plant Molecular Biology andBiotechnology 89-119 (CRC Press, 1993). In using the term, it is meantto include all types of selection markers, whether they be scorable orselective. Expression of such a nucleotide sequence can provide a meansfor selecting for a cell containing the construct, for example, byconferring a desirable phenotype to a plant cell containing thenucleotide sequence. For example, the additional nucleotide sequence canbe, or encode, a selectable marker, which, when present or expressed ina plant cell, provides a means to identify the plant cell containing themarker.

A selectable marker provides a means for screening a population oforganisms or cells of an organism (e.g., plants or plant cells) toidentify those having the marker and, therefore, the transgene ofinterest. A selectable marker generally confers a selective advantage tothe cell, or to an organism (e.g., a plant) containing the cell, forexample, the ability to grow in the presence of a negative selectiveagent such as an antibiotic or, for a plant, an herbicide. A selectiveadvantage also can be due, for example, to an enhanced or novel capacityto utilize an added compound as a nutrient, growth factor or energysource. A selective advantage can be conferred by a singlepolynucleotide, or its expression product, or by a combination ofpolynucleotides whose expression in a plant cell gives the cell apositive selective advantage, a negative selective advantage, or both.It should be recognized that expression of the transgene of interest(e.g., encoding a hpRNA) also provides a means to select cellscontaining the encoding nucleotide sequence. However, the use of anadditional selectable marker, which, for example, allows a plant cell tosurvive under otherwise toxic conditions, provides a means to enrich fortransformed plant cells containing the desired transgene. Examples ofsuitable scorable or selection genes known in the art can be found in,for example, Jefferson, et al., (1991) in Plant Molecular BiologyManual, ed. Gelvin, et al., (Kluwer Academic Publishers), pp. 1-33;DeWet, et al., (1987) Mol. Cell. Biol. 7:725-737; Goff, et al., (1990)EMBO J. 9:2517-2522; Kain, et al., (1995) BioTechniques 19:650-655; andChiu, et al., (1996) Curr. Biol. 6:325-330.

Examples of selectable markers include those that confer resistance toantimetabolites such as herbicides or antibiotics, for example,dihydrofolate reductase, which confers resistance to methotrexate(Reiss, (1994) Plant Physiol. (Life Sci. Adv.) 13:143-149; see also,Herrera-Estrella, et al., (1983) Nature 303:209-213; Meijer, et al.,(1991) Plant Mol. Biol. 16:807-820); neomycin phosphotransferase, whichconfers resistance to the aminoglycosides neomycin, kanamycin andparomycin (Herrera-Estrella, (1983) EMBO J. 2:987-995) and hygro, whichconfers resistance to hygromycin (Marsh, (1984) Gene 32:481-485; seealso, Waldron, et al., (1985) Plant Mol. Biol. 5:103-108; Zhijian, etal., (1995) Plant Science 108:219-227); trpB, which allows cells toutilize indole in place of tryptophan; hisD, which allows cells toutilize histinol in place of histidine (Hartman, (1988) Proc. Natl.Acad. Sci. USA 85:8047); mannose-6-phosphate isomerase which allowscells to utilize mannose (WO 94/20627); ornithine decarboxylase, whichconfers resistance to the ornithine decarboxylase inhibitor,2-(difluoromethyl)-DL-ornithine (DFMO; McConlogue, 1987, In: CurrentCommunications in Molecular Biology, Cold Spring Harbor Laboratory ed.);and deaminase from Aspergillus terreus, which confers resistance toBlasticidin S (Tamura, (1995) Biosci. Biotechnol. Biochem.59:2336-2338). Additional selectable markers include, for example, amutant EPSPV-synthase, which confers glyphosate resistance (Hinchee, etal., (1998) BioTechnology 91:915-922), a mutant acetolactate synthase,which confers imidazolinone or sulfonylurea resistance (Lee, et al.,(1988) EMBO J. 7:1241-1248), a mutant psbA, which confers resistance toatrazine (Smeda, et al., (1993) Plant Physiol. 103:911-917), or a mutantprotoporphyrinogen oxidase (see, U.S. Pat. No. 5,767,373), or othermarkers conferring resistance to an herbicide such as glufosinate.Examples of suitable selectable marker genes include, but are notlimited to, genes encoding resistance to chloramphenicol(Herrera-Estrella, et al., (1983) EMBO J. 2:987-992); streptomycin(Jones, et al., (1987) Mol. Gen. Genet. 210:86-91); spectinomycin(Bretagne-Sagnard, et al., (1996) Transgenic Res. 5:131-137); bleomycin(Hille, et al., (1990) Plant Mol. Biol. 7:171-176); sulfonamide(Guerineau, et al., (1990) Plant Mol. Biol. 15:127-136); bromoxynil(Stalker, et al., (1988) Science 242:419-423); glyphosate (Shaw, et al.,(1986) Science 233:478-481); phosphinothricin (DeBlock, et al., (1987)EMBO J. 6:2513-2518), and the like. One option for use of a selectivegene is a glufosinate-resistance encoding DNA and in one embodiment canbe the phosphinothricin acetyl transferase (“PAT”), maize optimized PATgene or bar gene under the control of the CaMV 35S or ubiquitinpromoters. The genes confer resistance to bialaphos. See, Gordon-Kamm,et al., (1990) Plant Cell 2:603; Uchimiya, et al., (1993) BioTechnology11:835; White, et al., (1990) Nucl Acids Res. 18:1062; Spencer, et al.,(1990) Theor. Appl. Genet. 79:625-631; and Anzai, et al., (1989) Mol.Gen. Gen. 219:492). A version of the PAT gene is the maize optimized PATgene, described at U.S. Pat. No. 6,096,947.

In addition, markers that facilitate identification of a plant cellcontaining the polynucleotide encoding the marker include, for example,luciferase (Giacomin, (1996) Plant Sci. 116:59-72; Scikantha, (1996) J.Bacteriol. 178:121), green fluorescent protein (Gerdes, (1996) FEBSLett. 389:44-47; Chalfie, et al., (1994) Science 263:802), and otherfluorescent protein variants, or β-glucuronidase (Jefferson, (1987)Plant Mol. Biol. Rep. 5:387; Jefferson, et al., (1987) EMBO J.6:3901-3907; Jefferson, (1989) Nature 342(6251):837-838); the maizegenes regulating pigment production (Ludwig, et al., (1990) Science247:449; Grotewold, et al., (1991) PNAS 88:4587-4591; Cocciolone, etal., (2001) Plant J 27(5):467-478; Grotewold, et al., (1998) Plant Cell10:721-740); β-galactosidase (Teeri, et al., (1989) EMBO J. 8:343-350);luciferase (Ow, et al., (1986) Science 234:856-859); chloramphenicolacetyltransferase (CAT) (Lindsey and Jones, (1987) Plant Mol. Biol.10:43-52); and numerous others as disclosed herein or otherwise known inthe art. Such markers also can be used as reporter molecules. Manyvariations on promoters, selectable markers and other components of theconstruct are available to one skilled in the art.

The term “plant” is used broadly herein to include any plant at anystage of development, or to part of a plant, including a plant cutting,a plant cell, a plant cell culture, a plant organ, a plant seed, and aplantlet. A plant cell is the structural and physiological unit of theplant, comprising a protoplast and a cell wall. A plant cell can be inthe form of an isolated single cell or aggregate of cells such as afriable callus, or a cultured cell, or can be part of a higher organizedunit, for example, a plant tissue, plant organ, or plant. Thus, a plantcell can be a protoplast, a gamete producing cell, or a cell orcollection of cells that can regenerate into a whole plant. As such, aseed, which comprises multiple plant cells and is capable ofregenerating into a whole plant, is considered a plant cell for purposesof this disclosure. A plant tissue or plant organ can be a seed,protoplast, callus, or any other groups of plant cells that is organizedinto a structural or functional unit. Particularly useful parts of aplant include harvestable parts and parts useful for propagation ofprogeny plants. A harvestable part of a plant can be any useful part ofa plant, for example, flowers, pollen, seedlings, tubers, leaves, stems,fruit, seeds, roots, and the like. A part of a plant useful forpropagation includes, for example, seeds, fruits, cuttings, seedlings,tubers, rootstocks, and the like.

A transgenic plant can be regenerated from a genetically modified plantcell, i.e., a whole plant can be regenerated from a plant cell; a groupof plant cells; a protoplast; a seed; or a piece of a plant such as aleaf, a cotyledon or a cutting. Regeneration from protoplasts variesamong species of plants. For example, a suspension of protoplasts can bemade and, in certain species, embryo formation can be induced from theprotoplast suspension, to the stage of ripening and germination. Theculture media generally contain various components necessary for growthand regeneration, including, for example, hormones such as auxins andcytokinins; and amino acids such as glutamic acid and proline, dependingon the particular plant species. Efficient regeneration will depend, inpart, on the medium, the genotype, and the history of the culture, andis reproducible if these variables are controlled.

Regeneration can occur from plant callus, explants, organs or plantparts. Transformation can be performed in the context of organ or plantpart regeneration. (see, Meth. Enzymol. Vol. 118; Klee, et al., (1987)Ann. Rev. Plant Physiol. 38:467). Utilizing the leafdisk-transformation-regeneration method, for example, disks are culturedon selective media, followed by shoot formation in about two to fourweeks (see, Horsch, et al., supra, 1985). Shoots that develop areexcised from calli and transplanted to appropriate root-inducingselective medium. Rooted plantlets are transplanted to soil as soon aspossible after roots appear. The plantlets can be repotted as required,until reaching maturity.

In seed-propagated crops, mature transgenic plants can beself-pollinated to produce a homozygous inbred plant. The resultinginbred plant produces seeds that contain the introduced transgene, andcan be grown to produce plants that express the polypeptide. Methods forbreeding plants and selecting for crossbred plants having desirablecharacteristics or other characteristics of interest include thosedisclosed herein and others well known to plant breeders.

In various aspects of the present invention, one or more transgenes isintroduced into cells. When used in reference to a transgene, the term“introducing” means transferring the exogenous nucleic acid moleculeinto a cell. A nucleic acid molecule can be introduced into a plant cellby a variety of methods. For example, the transgene can be contained ina vector, can be introduced into a plant cell using a direct genetransfer method such as electroporation or microprojectile mediatedtransformation, or using Agrobacterium mediated transformation. As usedherein, the term “transformed” refers to a plant cell containing anexogenously introduced nucleic acid molecule.

One or more exogenous nucleic acid molecules can be introduced intoplant cells using any of numerous well-known and routine methods forplant transformation, including biological and physical planttransformation protocols (see, e.g., Miki, et al., “Procedures forIntroducing Foreign DNA into Plants”; In Methods in Plant MolecularBiology and Biotechnology, Glick and Thompson, Eds. (CRC Press, Inc.,Boca Raton, 1993) pages 67-88). In addition, expression vectors and invitro culture methods for plant cell or tissue transformation andregeneration of plants are routine and well-known (see, e.g., Gruber, etal., “Vectors for Plant Transformation”; Id. at pages 89-119).

Suitable methods of transforming plant cells include microinjection,Crossway, et al., (1986) Biotechniques 4:320-334; electroporation,Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606;Agrobacterium-mediated transformation, see, for example, Townsend, etal., U.S. Pat. No. 5,563,055; direct gene transfer, Paszkowski, et al.,(1984) EMBO J. 3:2717-2722; and ballistic particle acceleration, see,for example, Sanford, et al., U.S. Pat. No. 4,945,050; Tomes, et al.,(1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods,ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe, et al.,(1988) Biotechnology 6:923-926. Also see, Weissinger, et al., (1988)Annual Rev. Genet. 22:421-477; Sanford, et al., (1987) ParticulateScience and Technology 5:27-37 (onion); Christou, et al., (1988) PlantPhysiol. 87:671-674 (soybean); McCabe, et al., (1988) Bio/Technology6:923-926 (soybean); Datta, et al., (1990) Biotechnology 8:736-740(rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein, et al., (1988) Biotechnology 6:559-563 (maize); Klein,et al., (1988) Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990)Biotechnology 8:833-839; Hooydaas-Van Slogteren, et al., (1984) Nature(London) 311:763-764; Bytebier, et al., (1987) Proc. Natl. Acad. Sci.USA 84:5345-5349 (Liliaceae); De Wet, et al., (1985) in The ExperimentalManipulation of Ovule Tissues, ed. G. P. Chapman, et al., (Longman, NewYork), pp. 197-209 (pollen); Kaeppler, et al., (1990) Plant Cell Reports9:415-418; and Kaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D. Halluin, et al., (1992) Plant Cell4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports12:250-255 and Christou, et al., (1995) Annals of Botany 75:407-413(rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maizevia Agrobacterium tumefaciens); all of which are herein incorporated byreference.

Agrobacterium-mediated transformation provides a useful method forintroducing a transgene into plants (Horsch, et al., (1985) Science227:1229 1985). A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria that genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant (see, e.g., Kado,(1991) Crit. Rev. Plant Sci. 10:1; see, also, Moloney, et al., (1989)Plant Cell Reports 8:238; U.S. Pat. No. 5,591,616; WO 99/47552;Weissbach and Weissbach, “Methods for Plant Molecular Biology” (AcademicPress, NY 1988), section VIII, pages 421-463; Grierson and Corey, “PlantMolecular Biology” 2d Ed. (Blackie, London 1988), Chapters 7-9; see,also, Horsch, et al., supra, 1985).

With respect to A. tumefaciens, the wild type form contains a Tiplasmid, which directs production of tumorigenic crown gall growth onhost plants. Transfer of the tumor-inducing T-DNA region of the Tiplasmid to a plant genome requires the Ti plasmid-encoded virulencegenes as well as T-DNA borders, which are a set of direct DNA repeatsthat delineate the region to be transferred. An Agrobacterium basedvector is a modified form of a Ti plasmid, in which the tumor-inducingfunctions are replaced by a nucleotide sequence of interest that is tobe introduced into the plant host. Methods of using Agrobacteriummediated transformation include cocultivation of Agrobacterium withcultured isolated protoplasts; transformation of plant cells or tissueswith Agrobacterium; and transformation of seeds, apices or meristemswith Agrobacterium. In addition, in planta transformation byAgrobacterium can be performed using vacuum infiltration of a suspensionof Agrobacterium cells (Bechtold, et al., (1993) C.R. Acad. Sci. Paris316:1194).

Agrobacterium-mediated transformation can employ cointegrate vectors orbinary vector systems, in which the components of the Ti plasmid aredivided between a helper vector, which resides permanently in theAgrobacterium host and carries the virulence genes, and a shuttlevector, which contains the gene of interest bounded by T-DNA sequences.Binary vectors are well known in the art (see, for example, De Framond,(1983) BioTechnology 1:262; Hoekema, et al., (1983) Nature 303:179) andare commercially available (Clontech; Palo Alto Calif.). Fortransformation, Agrobacterium can be cocultured, for example, with plantcells or wounded tissue such as leaf tissue, root explants, hypocotyls,cotyledons, stem pieces or tubers (see, for example, Glick and Thompson,“Methods in Plant Molecular Biology and Biotechnology” (Boca Raton Fla.,CRC Press 1993)). Wounded cells within the plant tissue that have beeninfected by Agrobacterium can develop organs de novo when cultured underthe appropriate conditions; the resulting transgenic shoots eventuallygive rise to transgenic plants which contain the introducedpolynucleotide.

Agrobacterium-mediated transformation has been used to produce a varietyof transgenic plants, including, for example, transgenic cruciferousplants such as Arabidopsis, mustard, rapeseed and flax; transgenicleguminous plants such as alfalfa, pea, soybean, trefoil and whiteclover; and transgenic solanaceous plants such as eggplant, petunia,potato, tobacco and tomato (see, for example, Wang, et al.,“Transformation of Plants and Soil Microorganisms” (Cambridge,University Press 1995)). In addition, Agrobacterium mediatedtransformation can be used to introduce an exogenous nucleic acidmolecule into apple, aspen, belladonna, black currant, carrot, celery,cotton, cucumber, grape, horseradish, lettuce, morning glory, muskmelon,neem, poplar, strawberry, sugar beet, sunflower, walnut, asparagus,rice, wheat, sorghum, barley, maize, and other plants (see, for example,Glick and Thompson, supra, 1993; Hiei, et al., (1994) Plant J.6:271-282; Shimamoto, (1995) Science 270:1772-1773).

Suitable strains of A. tumefaciens and vectors as well as transformationof Agrobacteria and appropriate growth and selection media are wellknown in the art (GV3101, pMK90RK), Koncz, (1986) Mol. Gen. Genet.204:383-396; (C58C1, pGV3850kan), Deblaere, (1985) Nucl. Acid Res.13:4777; Bevan, (1984) Nucleic Acid Res. 12:8711; Koncz, (1986) Proc.Natl. Acad. Sci. USA 86:8467-8471; Koncz, (1992) Plant Mol. Biol.20:963-976; Koncz, Specialized vectors for gene tagging and expressionstudies. In: Plant Molecular Biology Manual Vol. 2, Gelvin andSchilperoort (Eds.), Dordrecht, The Netherlands: Kluwer Academic Publ.(1994), 1-22; European Patent A-1 20 516; Hoekema: The Binary PlantVector System, Offsetdrukkerij Kanters B.V., Alblasserdam (1985),Chapter V; Fraley, Crit. Rev. Plant. Sci., 4:1-46; An, (1985) EMBO J.4:277-287).

As noted herein, the present invention provides vectors capable ofexpressing genes of interest under the control of the regulatoryelements. In general, the vectors should be functional in plant cells.At times, it may be preferable to have vectors that are functional in E.coli (e.g., production of protein for raising antibodies, DNA sequenceanalysis, construction of inserts, obtaining quantities of nucleicacids). Vectors and procedures for cloning and expression in E. coli arediscussed in Sambrook, et al., (supra).

The transformation vector, comprising the promoter of the presentinvention operably linked to an isolated nucleotide sequence in anexpression cassette, can also contain at least one additional nucleotidesequence for a gene to be co-transformed into the organism.Alternatively, the additional sequence(s) can be provided on anothertransformation vector.

Where the exogenous nucleic acid molecule is contained in a vector, thevector can contain functional elements, for example “left border” and“right border” sequences of the T-DNA of Agrobacterium, which allow forstable integration into a plant genome. Furthermore, methods and vectorsthat permit the generation of marker-free transgenic plants, forexample, where a selectable marker gene is lost at a certain stage ofplant development or plant breeding, are known, and include, forexample, methods of co-transformation (Lyznik, (1989) Plant Mol. Biol.13:151-161; Peng, (1995) Plant Mol. Biol. 27:91-104), or methods thatutilize enzymes capable of promoting homologous recombination in plants(see, e.g., W097/08331; Bayley, (1992) Plant Mol. Biol. 18:353-361;Lloyd, (1994) Mol. Gen. Genet. 242:653-657; Maeser, (1991) Mol. Gen.Genet. 230:170-176; Onouchi, (1991) Nucl. Acids Res. 19:6373-6378; see,also, Sambrook, et al., supra, 1989).

Direct gene transfer methods also can be used to introduce the desiredtransgene (or transgenes) into cells, including plant cells that arerefractory to Agrobacterium-mediated transformation (see, e.g., Hiei, etal., (1994) Plant J. 6:271-282; U.S. Pat. No. 5,591,616). Such methodsinclude direct gene transfer (see, European Patent Number A 164 575),injection, electroporation, biolistic methods such as particlebombardment, pollen-mediated transformation, plant RNA virus-mediatedtransformation, liposome-mediated transformation, transformation usingwounded or enzyme-degraded immature embryos, or wounded orenzyme-degraded embryogenic callus, and the like. Direct gene transfermethods include microprojectile-mediated (biolistic) transformationmethods, wherein the transgene is carried on the surface ofmicroprojectiles measuring 1 to 4 mm. A vector, particularly anexpression vector containing the transgene(s) of interest, is introducedinto plant tissues with a biolistic device that accelerates themicroprojectiles to speeds of 300 to 600 m/s, sufficient to penetrateplant cell walls and membranes (see, e.g., Sanford, et al., (1987) Part.Sci. Technol. 5:27; Sanford, (1988) Trends Biotech. 6:299, Klein, etal., (1988) BioTechnology 6:559-563; Klein, et al., (1992) BioTechnology10:268). In maize, for example, several target tissues can be bombardedwith DNA-coated microprojectiles in order to produce transgenic plants,including, for example, callus (Type I or Type II), immature embryos,and meristem tissue.

Other methods for physical delivery of a transgene into plants utilizesonication of the target cells (Zhang, et al., (1991) BioTechnology9:996); liposomes or spheroplast fusion (Deshayes, et al., (1985) EMBOJ. 4:2731; Christou, et al., (1987) Proc Natl. Acad. Sci., USA 84:3962);CaCl₂ precipitation or incubation with polyvinyl alcohol orpoly-L-ornithine (Hain, et al., (1985) Mol. Gen. Genet. 199:61; Draper,et al., (1982) Plant Cell Physiol. 23:451); and electroporation ofprotoplasts and whole cells and tissues (Donn, et al., (1990) In“Abstracts of VIIIth International Congress on Plant Cell and TissueCulture” IAPTC, A2-38, pg. 53; D'Halluin, et al., (1992) Plant Cell4:1495-1505; Spencer, et al., (1994) Plant Mol. Biol. 24:51-61).

A direct gene transfer method such as electroporation can beparticularly useful for introducing exogenous nucleic acid moleculesinto a cell such as a plant cell. For example, plant protoplasts can beelectroporated in the presence of a recombinant nucleic acid molecule,which can be in a vector (Fromm, et al., (1985) Proc. Natl. Acad. Sci.,USA 82:5824). Electrical impulses of high field strength reversiblypermeabilize membranes allowing the introduction of the nucleic acid.Electroporated plant protoplasts reform the cell wall, divide and form aplant callus. Microinjection can be performed as described in Potrykusand Spangenberg (eds.), Gene Transfer To Plants. Springer Verlag,Berlin, N.Y. (1995). A transformed plant cell containing the introducedrecombinant nucleic acid molecule can be identified due to the presenceof a selectable marker included in the construct.

As mentioned above, microprojectile mediated transformation alsoprovides a useful method for introducing exogenous nucleic acidmolecules into a plant cell (Klein et al., Nature 327:70-73, 1987). Thismethod utilizes microprojectiles such as gold or tungsten, which arecoated with the desired nucleic acid molecule by precipitation withcalcium chloride, spermidine or polyethylene glycol. The microprojectileparticles are accelerated at high speed into a plant tissue using adevice such as the BIOLISTIC PD-1000 particle gun (BioRad; HerculesCalif.). Microprojectile mediated delivery (“particle bombardment”) isespecially useful to transform plant cells that are difficult totransform or regenerate using other methods. Methods for thetransformation using biolistic methods are well known (Wan, (1984) PlantPhysiol. 104:37-48; Vasil, (1993) BioTechnology 11:1553-1558; Christou,(1996) Trends in Plant Science 1:423-431). Microprojectile mediatedtransformation has been used, for example, to generate a variety oftransgenic plant species, including cotton, tobacco, corn, wheat, oat,barley, sorghum, rice, hybrid poplar and papaya (see, Glick andThompson, supra, 1993; Duan, et al., (1996) Nature Biotech. 14:494-498;Shimamoto, (1994) Curr. Opin. Biotech. 5:158-162).

A rapid transformation regeneration system for the production oftransgenic plants such as a system that produces transgenic wheat in twoto three months (see, European Patent Number EP 0709462A2) also can beuseful for producing a transgenic plant according to a method of theinvention, thus allowing more rapid identification of gene functions.The transformation of most dicotyledonous plants is possible with themethods described above. Transformation of monocotyledonous plants alsocan be transformed using, for example, biolistic methods as describedabove, protoplast transformation, electroporation of partiallypermeabilized cells, introduction of DNA using glass fibers,Agrobacterium mediated transformation, and the like.

Plastid transformation also can be used to introduce a nucleic acidmolecule into a plant cell (U.S. Pat. Nos. 5,451,513, 5,545,817 and5,545,818; WO 95/16783; McBride, et al., (1994) Proc. Natl. Acad. Sci.,USA 91:7301-7305). Chloroplast transformation involves introducingregions of cloned plastid DNA flanking a desired nucleotide sequence,for example, a selectable marker together with polynucleotide ofinterest, into a suitable target tissue, using, for example, a biolisticor protoplast transformation method (e.g., calcium chloride or PEGmediated transformation). One to 1.5 kb flanking regions (“targetingsequences”) facilitate homologous recombination with the plastid genome,and allow the replacement or modification of specific regions of theplastome. Using this method, point mutations in the chloroplast 16S rRNAand rps12 genes, which confer resistance to spectinomycin andstreptomycin and can be utilized as selectable markers fortransformation (Svab, et al., (1990) Proc. Natl. Acad. Sci., USA87:8526-8530; Staub and Maliga, (1992) Plant Cell 4:39-45), resulted instable homopiasmic transformants, at a frequency of approximately oneper 100 bombardments of target leaves. The presence of cloning sitesbetween these markers allowed creation of a plastid targeting vector forintroduction of foreign genes (Staub and Maliga, (1993) EMBO J.12:601-606). Substantial increases in transformation frequency areobtained by replacement of the recessive rRNA or r-protein antibioticresistance genes with a dominant selectable marker, the bacterial aadAgene encoding the spectinomycin-detoxifying enzymeaminoglycoside-3′-adenyltransferase (Svab and Maliga, (1993) Proc. Natl.Acad. Sci., USA 90:913-917). Approximately 15 to 20 cell division cyclesfollowing transformation are generally required to reach a homoplastidicstate. Plastid expression, in which genes are inserted by homologousrecombination into all of the several thousand copies of the circularplastid genome present in each plant cell, takes advantage of theenormous copy number advantage over nuclear-expressed genes to permitexpression levels that can readily exceed 10% of the total soluble plantprotein.

The cells that have been transformed can be grown into plants inaccordance with conventional ways. See, for example, McCormick, et al.,(1986) Plant Cell Reports 5:81-84. These plants can then be grown andpollinated with the same transformed strain or different strains, andresulting plants having expression of the desired phenotypiccharacteristic can then be identified. Two or more generations can begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited.

Plants suitable for purposes of the present invention can be monocots ordicots and include, but are not limited to, maize, wheat, barley, rye,sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower,broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper,celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon,plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry,blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato,sorghum, sugarcane, sugar beet, sunflower, rapeseed, clover, tobacco,carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsisthaliana, and woody plants such as coniferous and deciduous trees. Thus,a transgenic plant or genetically modified plant cell of the inventioncan be an angiosperm or gymnosperm.

Angiosperms are divided into two broad classes based on the number ofcotyledons, which are seed leaves that generally store or absorb food; amonocotyledonous angiosperm has a single cotyledon, and a dicotyledonousangiosperm has two cotyledons. Angiosperms produce a variety of usefulproducts including materials such as lumber, rubber, and paper; fiberssuch as cotton and linen; herbs and medicines such as quinine andvinblastine; ornamental flowers such as roses and, where included withinthe scope of the present invention, orchids; and foodstuffs such asgrains, oils, fruits and vegetables. Angiosperms encompass a variety offlowering plants, including, for example, cereal plants, leguminousplants, oilseed plants, hardwood trees, fruit-bearing plants andornamental flowers, which general classes are not necessarily exclusive.Cereal plants, which produce an edible grain, include, for example,corn, rice, wheat, barley, oat, rye, orchardgrass, guinea grass, andsorghum. Leguminous plants include members of the pea family (Fabaceae)and produce a characteristic fruit known as a legume. Examples ofleguminous plants include, for example, soybean, pea, chickpea, mothbean, broad bean, kidney bean, lima bean, lentil, cowpea, dry bean, andpeanut, as well as alfalfa, birdsfoot trefoil, clover and sainfoin.Oilseed plants, which have seeds that are useful as a source of oil,include soybean, sunflower, rapeseed (canola) and cottonseed.Angiosperms also include hardwood trees, which are perennial woodyplants that generally have a single stem (trunk). Examples of such treesinclude alder, ash, aspen, basswood (linden), beech, birch, cherry,cottonwood, elm, eucalyptus, hickory, locust, maple, oak, persimmon,poplar, sycamore, walnut, sequoia, and willow. Trees are useful, forexample, as a source of pulp, paper, structural material and fuel.

Angiosperms produce seeds enclosed within a mature, ripened ovary. Anangiosperm fruit can be suitable for human or animal consumption or forcollection of seeds to propagate the species. For example, hops are amember of the mulberry family that are prized for their flavoring inmalt liquor. Fruit-bearing angiosperms also include grape, orange,lemon, grapefruit, avocado, date, peach, cherry, olive, plum, coconut,apple and pear trees and blackberry, blueberry, raspberry, strawberry,pineapple, tomato, cucumber and eggplant plants. An ornamental flower isan angiosperm cultivated for its decorative flower. Examples ofcommercially important ornamental flowers include rose, lily, tulip andchrysanthemum, snapdragon, camellia, carnation and petunia plants, andcan include orchids. It will be recognized that the present inventionalso can be practiced using gymnosperms, which do not produce seeds in afruit.

Certain embodiments of this invention overcome the problem ofmaintenance of homozygous recessive reproductive traits when using atransgenic restoration approach, while decreasing the number of plants,plantings and steps needed for maintenance of plants with such traits.

Homozygosity is a genetic condition existing when identical allelesreside at corresponding loci on homologous chromosomes. Heterozygosityis a genetic condition existing when different alleles reside atcorresponding loci on homologous chromosomes. Hemizygosity is a geneticcondition existing when there is only one copy of a gene (or set ofgenes) with no allelic counterpart on the sister chromosome.

Maintenance of the homozygous recessive condition for male sterility isachieved by introducing into a plant a restoration transgene constructthat is linked to a sequence which interferes with the formation,function, or dispersal of male gametes of the plant, to create a“maintainer” or “donor” plant. The restoring transgene, uponintroduction into a plant that is homozygous recessive for the malesterility genetic trait, restores the genetic function of that trait.Due to the linked gene driven by a male-gamete-specific-promoter, allpollen containing the restoration transgene is rendered nonviable. Allviable pollen produced contains a copy of the recessive allele but doesnot contain the restoration transgene. The transgene is kept in thehemizygous state in the maintainer plant.

The pollen from the maintainer can be used to fertilize plants that arehomozygous for the recessive trait, and the progeny will thereforeretain their homozygous recessive condition. The maintainer plantcontaining the restoring transgene construct is propagated byself-fertilization, with half of the resulting seed used to producefurther plants that are homozygous recessive for the gene of interestand hemizygous for the restoring transgene construct.

The maintainer plant serves as a pollen donor to the plant having thehomozygous recessive trait. The maintainer is optimally produced from aplant having the homozygous recessive trait and which also hasnucleotide sequences introduced therein which would restore the traitcreated by the homozygous recessive alleles. Further, the restorationsequence is linked to nucleotide sequences that interfere with thefunction, formation, or dispersal of male gametes. The gene can operateto prevent formation of male gametes or prevent function of the malegametes by any of a variety of well-known modalities and is not limitedto a particular methodology. By way of example but not limitation, thiscan include use of one or more genes which express a product cytotoxicto male gametes (see, for example, U.S. Pat. Nos. 5,792,853; and5,689,049; PCT/EP89/00495); inhibit product formation of another geneimportant to male gamete formation, function, or dispersal (see, U.S.Pat. Nos. 5,859,341 and 6,297,426); combine with another gene product toproduce a substance preventing gamete formation, function, or dispersal(see, U.S. Pat. Nos. 6,162,964; 6,013,859; 6,281,348; 6,399,856;6,248,935; 6,750,868 and 5,792,853); are antisense to or causeco-suppression of a gene critical to male gamete formation, function, ordispersal (see, U.S. Pat. Nos. 6,184,439; 5,728,926; 6,191,343;5,728,558 and 5,741,684), or the like.

Ordinarily, to produce more plants having the recessive condition, onemight cross the recessive plant with another recessive plant, or selfpollinate a recessive plant. This may not be desirable for somerecessive traits and may be impossible for recessive traits affectingreproductive development. Alternatively, one could cross the homozygousplant with a second plant having the restoration gene, but this requiresfurther crossing to segregate away the restoring gene to once againreach the recessive phenotypic state. Instead, in one embodiment theinvention provides a process in which the homozygous recessive conditioncan be maintained, while crossing it with the maintainer plant. Thismethod can be used with any situation in which it is desired to continuethe recessive condition. This results in a relatively simple,cost-effective system for maintaining a population of homozygousrecessive plants.

When the homozygous recessive condition is one that produces malesterility, the maintainer plant, of necessity, must contain a functionalrestoring transgene construct capable of complementing the mutation andrendering the homozygous recessive plant able to produce viable pollen.Linking this male fertility restoration gene with a second functionalnucleotide sequence which interferes with the formation, function, ordispersal of the male gametes of the plant results in a maintainer plantthat produces pollen containing only the recessive allele of therestored gene at its native locus due to the pollen-specific cytotoxicaction of the second nucleotide sequence. This viable pollen fraction isnon-transgenic with regard to the restoring transgene construct.

For example, it is desirable to produce male sterile female plants foruse in the hybrid production process which are sterile as a result ofbeing homozygous for a mutation in the MS45 gene, a gene which isessential for male fertility. Such a mutant MS45 allele is designated asms45. A plant that is homozygous for ms45 (represented by the notationms45/ms45) displays the homozygous recessive male sterility phenotypeand produces no functional pollen. See, U.S. Pat. Nos. 5,478,369;5,850,014; 6,265,640 and 5,824,524. In both the inbred and hybridproduction processes, it is highly desired to maintain this homozygousrecessive condition. When sequences encoding the MS45 gene areintroduced into a plant having the homozygous condition, sporophyticrestoration of male fertility results. (Cigan, et al., (2001) Sex. PlantRepro. 14:135-142). By the method of the invention, a plant which isms45/ms45 homozygous recessive may have introduced into it a functionalMS45 gene, and thus male fertility is restored. This gene can be linkedto a second gene which operates to render pollen nonfunctional or whichprevents its formation, or which produces a lethal product in pollen,and which is linked to a promoter directing its expression in the malegametes. This results in a plant which produces viable pollen containingms45 without the restoring transgene construct.

An example is a construct that includes the MS45 gene operably linked tothe 5126 promoter, a male tissue-preferred promoter (see, U.S. Pat. No.5,837,851) and further linked to the cytotoxic DAM methylase gene undercontrol of the PG47 promoter (see, U.S. Pat. Nos. 5,792,853 and5,689,049). The resulting plant produces pollen, but the only viablepollen contains the ms45 gene. It can therefore be used as a pollinatorto fertilize the homozygous recessive plant (ms45/ms45), and 100% of theprogeny produced will continue to be male sterile as a result ofmaintaining homozygosity for ms45. The progeny will not contain theintroduced restoring transgene construct.

Clearly, many variations on this method are available as it relates tomale sterility. Any other gene critical to male fertility may be used inthis system. For example and without limitation, such genes can includethe SBMu200 gene (also known as SB200 or MS26) described at WO 02/26789;the BS92-7 gene (also known as BS7) described at WO 02/063021; MS2 genedescribed at Albertsen and Phillips, “Developmental Cytology of 13Genetic Male Sterile Loci in Maize” Canadian Journal of Genetics &Cytology 23:195-208 (January 1981); or the Arabadopsis MS2 genedescribed at Aarts et al., “Transposon Tagging of a Male Sterility Genein Arabidopsis”, Nature, 363:715-717 (Jun. 24, 1993); and theArabidopsis gene MS1 described at Wilson, et al., “The Arabidopsis MALESTERILITY1 (MS1) gene is a transcriptional regulator of malegametogenesis, with homology to the PHD-finger family of transcriptionfactors”, Plant J., 1:27-39 (Oct. 28, 2001).

A desirable result of the process of the invention is that the planthaving the restorer nucleotide sequence may be self-fertilized; that is,pollen from the plant transferred to the flower of the same plant toachieve the propagation of restorer plants. (Note that “selffertilization” includes both the situation where the plant producing thepollen is fertilized with that same pollen, and the situation wherepollen from a plant, or from a group of genetically identical plants,pollinates a plant which is a genetically identical individual, or agroup of such genetically identical plants.) The restoring transgeneconstruct will not be present in the pollen, but it will be contained in50% of the ovules (the female gamete). The seed resulting from theself-fertilization can be planted, and selection made for the seedhaving the restoring transgene construct. The selection process canoccur by any one or more of many known processes, the most common beingwhere the restoration nucleotide sequence is linked to a marker gene.The marker can be scorable or selectable, and allows identification ofthe seed comprising the restoration sequence, and/or of those plantsproduced from the seed having the restoration sequence.

In an embodiment of the invention, it is possible to provide that thepromoter driving the restoration gene is inducible. Additional controlis thus allowed in the process, where so desired, by providing that theplant having the restoration nucleotide sequences is constitutively malesterile. This type of male sterility is set forth the in U.S. Pat. No.5,859,341. In order for the plant to become fertile, the inducingsubstance must be provided, and the plant will become fertile. Again,when combined with the process of the invention as described supra, theonly pollen produced will not contain the restoration nucleotidesequences.

In yet another embodiment of the invention, the gamete controlling thetransmission of the restoration nucleotide sequences can be the femalegamete, instead of the male gamete. The process is the same as thatdescribed above, with the exception in those instances where one alsodesires to maintain the plant having the restoration nucleotidesequences by self fertilization. In that case, it will be useful toprovide that the promoter driving the restoration gene is inducible, sothat female fertility may be triggered by exposure to the inducingsubstance, and seed can be formed. Control of female fertility in such amanner is described at U.S. Pat. No. 6,297,426. Examples of genesimpacting female fertility include the teosinte branched1 (Tb1) gene,which increases apical dominance, resulting in multiple tassels andrepression of female tissue. Hubbard, et al., (2002) Genetics162:1927-1935; Doebley, et al., (1997) Nature 386:485-488 (1997).Another example is the so-called “barren 3” or “ba3”. This mutant wasisolated from a mutant maize plant infected with wheat-streak mosaicvirus and is described at Pan and Peterson, (1992) J. Genet. And Breed.46:291-294. The plants develop normal tassels but do not have any earshoots along the stalks. Barren-stalk fastigiate is described at Coe andBeckett, (1987) Maize Genet. Coop. Newslett. 61:46-47. Other examplesinclude the barren stalk1 gene (Gallavotti, et al., (2004) Nature432:630-635); lethal ovule mutant (Vollbrecht, (1994) Maize GeneticsCooperation Newsletter 68:2-3); and defective pistil mutant (Miku andMustyatsa, (1978) Genetika 14(2):365-368).

Any plant-compatible promoter elements can be employed to controlexpression of the regions of the restoring transgene construct thatencode specific proteins and functions. Those can be plant genepromoters, such as, for example, the ubiquitin promoter, the promoterfor the small subunit of ribulose-1,5-bis-phosphate carboxylase, orpromoters from the tumor-inducing plasmids from Agrobacteriumtumefaciens, such as the nopaline synthase and octopine synthasepromoters, or viral promoters such as the cauliflower mosaic virus(CaMV) 19S and 35S promoters or the figwort mosaic virus 35S promoter.See, Kay, et al., (1987) Science 236:1299 and European PatentApplication Number 0 342 926. See, international application number WO91/19806 for a review of illustrative plant promoters suitably employedin the present invention. The range of available plant-compatiblepromoters includes tissue-specific and inducible promoters.

The invention contemplates the use of promoters providingtissue-preferred expression, including promoters which preferentiallyexpress to the gamete tissue, male or female, of the plant. Theinvention does not require that any particular gamete tissue-preferredpromoter be used in the process, and any of the many such promotersknown to one skilled in the art may be employed. By way of example, butnot limitation, one such promoter is the 5126 promoter, whichpreferentially directs expression of the gene to which it is linked tomale tissue of the plants, as described in U.S. Pat. Nos. 5,837,851 and5,689,051. Other examples include the MS45 promoter described at U.S.Pat. No. 6,037,523; SF3 promoter described at U.S. Pat. No. 6,452,069;the BS92-7 or BS7 promoter described at WO 02/063021; the SBMu200promoter described at WO 02/26789; a SGB6 regulatory element describedat U.S. Pat. No. 5,470,359, and TA39 (Koltunow, et al., (1990)“Different temporal and spatial gene expression patterns occur duringanther development.” Plant Cell 2:1201-1224; Goldberg, et al., (1993)Anther development: basic principles and practical applications. PlantCell 5:1217-1229; and U.S. Pat. No. 6,399,856. See also, Nadeau, et al.,(1996) Plant Cell 8(2):213-39; and Lu, et al., (1996) Plant Cell8(12):2155-68.

The P67 promoter set forth in SEQ ID NO: 1 is 1112 nucleotides inlength. This promoter was isolated from a genomic clone corresponding toa maize EST sequence. The sequence showed limited homology to putativepectin methylesterase.

The pollen specificity of expression of P67 has been confirmed by RT-PCRand Northern blot analyses of RNA samples from different tissuesincluding leaf, root, anther/mature pollen grains, tassel at vacuolestage, spikelet, cob, husk, silk and embryo. The results indicate a highlevel of specificity for expression in developing pollen, particularlyat the mid-uninucleate stage.

Southern blot analysis has shown that the clone represents single- orlow-copy genes in the corn genome. Chromosome mapping using the oatchromosome substitution line revealed that the sequence is located atChromosome 1 of maize.

The clone was used to screen a maize BAC library. Positive BAC cloneshave been found and subcloned into pBluescript KS. Subclonescorresponding to the cDNA sequences have been identified and sequenced.The transcriptional start site has been determined using a RNAligase-mediated rapid amplification of 5′ end approach. The promoterregion was named P67.

The P95 promoter set forth in SEQ ID NO: 2 is 1092 nucleotides inlength. This promoter was isolated from a genomic clone corresponding toa maize EST sequence. The sequence showed limited homology to putativeL-ascorbate oxidase.

The pollen specificity of expression of P95 has been confirmed by RT-PCRand Northern blot analyses of RNA samples from different tissuesincluding leaf, root, anther/mature pollen grains, tassel at vacuolestage, spikelet, cob, husk, silk and embryo. The results indicate a highlevel of specificity for expression in developing pollen, particularlyat the mid-uninucleate stage.

Southern blot analysis has shown that the clone represents single- orlow-copy genes in the corn genome. Chromosome mapping using the oatchromosome substitution line revealed that the sequence is located atChromosomes 6 and 8 of maize.

The clone was used to screen a maize BAC library. Positive BAC cloneshave been found and subcloned into pBluescript KS. Subclonescorresponding to the cDNA sequences have been identified and sequenced.The transcriptional start site has been determined using a RNAligase-mediated rapid amplification of 5′ end approach. The promoterregion was named P95.

Using well-known techniques, additional promoter sequences may beisolated based on their sequence homology to SEQ ID NO: 1 or SEQ ID NO:2. In these techniques, all or part of a known promoter sequence is usedas a probe which selectively hybridizes to other sequences present in apopulation of cloned genomic DNA fragments (i.e. genomic libraries) froma chosen organism. Methods that are readily available in the art for thehybridization of nucleic acid sequences may be used to obtain sequenceswhich correspond to these promoter sequences in species including, butnot limited to, maize (corn; Zea mays), canola (Brassica napus, Brassicarapa ssp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secalecereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower(Helianthus annuus), wheat (Triticum aestivum), soybean (Glycine max),tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoeabatatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut(Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrusspp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musaspp.), avocado (Persea americana), fig (Ficus casica), guava (Psidiumguajava), mango (Mangifera indica), olive (Olea europaea), oats, barley,vegetables, ornamentals, and conifers. Preferably, plants include maize,soybean, sunflower, safflower, canola, wheat, barley, rye, alfalfa, andsorghum.

The entire promoter sequence or portions thereof can be used as a probecapable of specifically hybridizing to corresponding promoter sequences.To achieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique and are preferably at leastabout 10 nucleotides in length, and most preferably at least about 20nucleotides in length. Such probes can be used to amplify correspondingpromoter sequences from a chosen organism by the well-known process ofpolymerase chain reaction (PCR). This technique can be used to isolateadditional promoter sequences from a desired organism or as a diagnosticassay to determine the presence of the promoter sequence in an organism.Examples include hybridization screening of plated DNA libraries (eitherplaques or colonies; see e.g., Innis, et al., (1990) PCR Protocols, AGuide to Methods and Applications, eds., Academic Press).

In general, sequences that correspond to a promoter sequence of thepresent invention and hybridize to a promoter sequence disclosed hereinwill be at least 50% homologous, 55% homologous, 60% homologous, 65%homologous, 70% homologous, 75% homologous, 80% homologous, 85%homologous, 90% homologous, 95% homologous and even 98% homologous ormore with the disclosed sequence.

Fragments of a particular promoter sequence disclosed herein may operateto promote the pollen-preferred expression of an operably-linkedisolated nucleotide sequence. These fragments will comprise at leastabout 20 contiguous nucleotides, preferably at least about 50 contiguousnucleotides, more preferably at least about 75 contiguous nucleotides,even more preferably at least about 100 contiguous nucleotides of theparticular promoter nucleotide sequences disclosed herein. Thenucleotides of such fragments will usually comprise the TATA recognitionsequence of the particular promoter sequence. Such fragments can beobtained by use of restriction enzymes to cleave the naturally-occurringpromoter sequences disclosed herein; by synthesizing a nucleotidesequence from the naturally-occurring DNA sequence; or through the useof PCR technology. See particularly, Mullis, et al., (1987) MethodsEnzymol. 155:335-350, and Erlich, ed. (1989) PCR Technology (StocktonPress, New York). Again, variants of these fragments, such as thoseresulting from site-directed mutagenesis, are encompassed by thecompositions of the present invention.

Thus, nucleotide sequences comprising at least about 20 contiguousnucleotides of the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2are encompassed. These sequences can be isolated by hybridization, PCR,and the like. Such sequences encompass fragments capable of drivingpollen-preferred expression, fragments useful as probes to identifysimilar sequences, as well as elements responsible for temporal ortissue specificity.

Biologically active variants of the promoter sequence are alsoencompassed by the compositions of the present invention. A regulatory“variant” is a modified form of a promoter wherein one or more baseshave been modified, removed or added. For example, a routine way toremove part of a DNA sequence is to use an exonuclease in combinationwith DNA amplification to produce unidirectional nested deletions ofdouble-stranded DNA clones. A commercial kit for this purpose is soldunder the trade name Exo-Size™ (New England Biolabs, Beverly, Mass.).Briefly, this procedure entails incubating exonuclease III with DNA toprogressively remove nucleotides in the 3′ to 5′ direction at 5′overhangs, blunt ends or nicks in the DNA template. However, exonucleaseIII is unable to remove nucleotides at 3′, 4-base overhangs. Timeddigests of a clone with this enzyme produce unidirectional nesteddeletions.

One example of a regulatory sequence variant is a promoter formed bycausing one or more deletions in a larger promoter. Deletion of the 5′portion of a promoter up to the TATA box near the transcription startsite may be accomplished without abolishing promoter activity, asdescribed by Zhu, et al., (1995) The Plant Cell 7:1681-89. Such variantsshould retain promoter activity, particularly the ability to driveexpression in specific tissues. Biologically active variants include,for example, the native regulatory sequences of the invention having oneor more nucleotide substitutions, deletions or insertions. Activity canbe measured by Northern blot analysis, reporter activity measurementswhen using transcriptional fusions, and the like. See, for example,Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2nd ed.Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), hereinincorporated by reference.

The nucleotide sequences for the pollen-preferred promoters disclosed inthe present invention, as well as variants and fragments thereof, areuseful in the genetic manipulation of any plant when operably linkedwith an isolated nucleotide sequence whose expression is to becontrolled to achieve a desired phenotypic response.

The nucleotide sequence operably linked to the regulatory elementsdisclosed herein can be an antisense sequence for a targeted gene. By“antisense DNA nucleotide sequence” is intended a sequence that is ininverse orientation to the 5′-to-3′ normal orientation of thatnucleotide sequence. When delivered into a plant cell, expression of theantisense DNA sequence prevents normal expression of the DNA nucleotidesequence for the targeted gene. The antisense nucleotide sequenceencodes an RNA transcript that is complementary to and capable ofhybridizing with the endogenous messenger RNA (mRNA) produced bytranscription of the DNA nucleotide sequence for the targeted gene. Inthis case, production of the native protein encoded by the targeted geneis inhibited to achieve a desired phenotypic response. Thus theregulatory sequences claimed herein can be operably linked to antisenseDNA sequences to reduce or inhibit expression of a native or exogenousprotein in the plant.

Many nucleotide sequences are known which inhibit pollen formation orfunction or dispersal, and any sequences which accomplish thisinhibition will suffice. A discussion of genes which can impact properdevelopment or function is included at U.S. Pat. No. 6,399,856 andincludes dominant negative genes such as cytotoxin genes, methylasegenes, and growth-inhibiting genes. Dominant negative genes includediphtheria toxin A-chain gene (Czako and An (1991) Plant Physiol.95:687-692); cell cycle division mutants such as CDC in maize(Colasanti, et al., (1991) Proc. Natl. Acad. Sci. USA 88:3377-3381); theWT gene (Farmer, et al., (1994) Hum. Mol. Genet. 3:723-728); and P68(Chen, et al., (1991) Proc. Natl. Acad. Sci. USA 88:315-319). A suitablegene may also encode a protein involved in inhibiting pistildevelopment, pollen stigma interactions, pollen tube growth orfertilization, or a combination thereof. In addition, genes that eitherinterfere with the normal accumulation of starch in pollen or affectosmotic balance within pollen may also be suitable. These may include,for example, the maize alpha-amylase gene, maize beta-amylase gene,debranching enzymes such as Sugary1 and pullulanase, glucanase, andSacB.

In an illustrative embodiment, the DAM-methylase gene, the expressionproduct of which catalyzes methylation of adenine residues in the DNA ofthe plant, is used. Methylated adenines will not affect cell viabilityand will be found only in the tissues in which the DAM-methylase gene isexpressed, because such methylated residues are not found endogenouslyin plant DNA. Examples of so-called “cytotoxic” genes are discussedsupra and can include, but are not limited to pectate lyase gene pelE,from Erwinia chrysanthermi (Kenn, et al., (1986) J. Bacteriol 168:595);diphtheria toxin A-chain gene (Greenfield, et al., (1983) Proc. Natl.Acad. Sci. USA 80:6853, Palmiter, et al., (1987) Cell 50:435); T-urf13gene from cms-T maize mitochondrial genomes (Braun, et al., (1990) PlantCell 2:153; Dewey, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5374);CytA toxin gene from Bacillus thuringiensis Israeliensis that causescell membrane disruption (McLean, et al., (1987) J. Bacteriol 169:1017,U.S. Pat. No. 4,918,006); DNAses, RNAses, (U.S. Pat. No. 5,633,441);proteases, or genes expressing anti-sense RNA.

Further, the methods of the invention are useful in retaining thehomozygous recessive condition of traits other than those impactingplant fertility. The gene of interest which restores the condition wouldbe introduced into a plant linked to a nucleotide sequence whichinhibits the formation, function, or dispersal of pollen and which maybe further linked to a male gamete tissue-preferred promoter and a geneencoding a marker, for example a seed-specific marker. Viable pollenproduced by the plant into which the construct is introduced containsonly the recessive allele of the gene of interest and none of therestoring transgene sequences. Half of the female gametes of thehemizygous transgenic plant contain the transgene and can beself-pollinated, or pollinated by a plant comprising the recessivealleles. Half of the seeds produced will carry the transgene and can beidentified by means of the linked marker. The hemizygous condition canbe maintained by selfing the hemizygous plant; half of the offspringwill contain the transgene and thus the trait of interest.

Genes of interest are reflective of the commercial markets and interestsof those involved in the development of the crop. Crops and markets ofinterest change, and as developing nations open up world markets, newcrops and technologies will emerge also. In addition, as ourunderstanding of agronomic traits and characteristics such as yield andheterosis increases, the choice of genes for transformation will changeaccordingly.

Regulation of male fertility is necessarily measured in terms of itseffect on individual cells. For example, suppression in 99.99% of pollengrains is required to achieve reliable sterility for commercial use.However, successful suppression or restoration of expression of othertraits may be accomplished with lower stringency. Within a particulartissue, for example, expression in 98%, 95%, 90%, 80% or fewer cells mayresult in the desired phenotype.

This invention has utility for a variety of recessive genes, not limitedto those where expression of the homozygous recessive trait compromisesthe plant's ability to maintain its full reproductive capacity. Generalcategories of genes of interest include, for example, those genesinvolved in information, such as zinc fingers, those involved incommunication, such as kinases, and those involved in housekeeping, suchas heat shock proteins. More specific categories of transgenes, forexample, include genes encoding important traits for agronomics, insectresistance, disease resistance, herbicide resistance, sterility, graincharacteristics, and commercial products. Genes of interest include,generally, those involved in oil, starch, carbohydrate, or nutrientmetabolism as well as those affecting kernel size, sucrose loading, andthe like. Agronomically important traits such as oil, starch, andprotein content can be genetically altered in addition to usingtraditional breeding methods. Modifications include increasing contentof oleic acid, saturated and unsaturated oils, increasing levels oflysine and sulfur, providing essential amino acids, and alsomodification of starch. Hordothionin protein modifications are describedin U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802 and 5,990,389. Anotherexample is lysine and/or sulfur rich seed protein encoded by the soybean2S albumin described in U.S. Pat. No. 5,850,016, and the chymotrypsininhibitor from barley, described in Williamson, et al., (1987) Eur. J.Biochem. 165:99-106. Other important genes encode growth factors andtranscription factors.

Agronomic traits can be improved by altering expression of genes that:affect growth and development, especially during environmental stress.These include, for example, genes encoding cytokinin biosynthesisenzymes, such as isopentenyl transferase; genes encoding cytokinincatabolic enzymes, such as cytokinin oxidase; genes encodingpolypeptides involved in regulation of the cell cycle, such as CyclinDor cdc25; genes encoding cytokinin receptors or sensors, such as CRE1,CKI1, and CKI2, histidine phospho-transmitters, or cytokinin responseregulators.

Insect resistance genes may encode resistance to pests that have greatyield drag such as rootworm, cutworm, European Corn Borer, and the like.Such genes include, for example: Bacillus thuringiensis endotoxin genes,U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756 and 5,593,881;Geiser, et al., (1986) Gene 48:109; lectins, Van Damme, et al., (1994)Plant Mol. Biol. 24:825; and the like.

Genes encoding disease resistance traits include: detoxification genes,such as against fumonisin (WO 9606175 filed Jun. 7, 1995); avirulence(avr) and disease resistance (R) genes, Jones, et al., (1994) Science266:789; Martin, et al., (1993) Science 262:1432; Mindrinos, et al.,(1994) Cell 78:1089; and the like.

Commercial traits can also be encoded on a gene(s) which could alter orincrease for example, starch for the production of paper, textiles andethanol, or provide expression of proteins with other commercial uses.Another important commercial use of transformed plants is the productionof polymers and bioplastics such as described in U.S. Pat. No. 5,602,321issued Feb. 11, 1997. Genes such as B-Ketothiolase, PHBase(polyhydroxybutyrate synthase) and acetoacetyl-CoA reductase (see,Schubert, et al., (1988) J. Bacteriol 170(12):5837-5847) facilitateexpression of polyhydroxyalkanoates (PHAs).

Exogenous products include plant enzymes and products as well as thosefrom other sources including prokaryotes and other eukaryotes. Suchproducts include enzymes, cofactors, hormones, and the like. The levelof seed proteins, particularly modified seed proteins having improvedamino acid distribution to improve the nutrient value of the seed, canbe increased. This is achieved by the expression of such proteins havingenhanced amino acid content.

Expression cassettes of the invention, comprising a promoter andisolated nucleotide sequence of interest, may also include, at the 3′terminus of the isolated nucleotide sequence of interest, atranscriptional and translational termination region functional inplants. The termination region can be native with the promoternucleotide sequence of the cassette, can be native with the DNA sequenceof interest, or can be derived from another source.

Other convenient termination regions are available from the Ti-plasmidof A. tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also, Guerineau, et al., (1991) Mol. Gen.Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon, et al.,(1991) Genes Dev. 5:141-149; Mogen, et al., (1990) Plant Cell2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Ballas, et al.,1989) Nucleic Acids Res. 17:7891-7903; Joshi, et al., (1987) NucleicAcid Res. 15:9627-9639.

The expression cassettes can additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample: EMCV leader (Encephalomyocarditis 5′ noncoding region),Elroy-Stein, et al., (1989) Proc. Nat. Acad. Sci. USA 86:6126-6130;potyvirus leaders, for example, TEV leader (Tobacco Etch Virus),Allison, et al., (1986); MDMV leader (Maize Dwarf Mosaic Virus),Virology 154:9-20; human immunoglobulin heavy-chain binding protein(BiP), Macejak, et al., (1991) Nature 353:90-94; untranslated leaderfrom the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), Jobling,et al., (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV),Gallie, et al., (1989) Molecular Biology of RNA, pages 237-256; andmaize chlorotic mottle virus leader (MCMV) Lommel, et al., (1991)Virology 81:382-385. See also, Della-Cioppa, et al., (1987) PlantPhysiology 84:965-968. The cassette can also contain sequences thatenhance translation and/or mRNA stability such as introns.

In those instances where it is desirable to have the expressed productof the isolated nucleotide sequence directed to a particular organelle,particularly the plastid, amyloplast, or to the endoplasmic reticulum,or secreted at the cell's surface or extracellularly, the expressioncassette can further comprise a coding sequence for a transit peptide.Such transit peptides are well known in the art and include, but are notlimited to: the transit peptide for the acyl carrier protein, the smallsubunit of RUBISCO, plant EPSP synthase, and the like.

In preparing the expression cassette, the various DNA fragments can bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers can be employed to join the DNA fragmentsor other manipulations can be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction digests, annealing, and resubstitutions such astransitions and transversions, can be involved.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “percentage of sequenceidentity”, and (d) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, a segment of afull-length promoter sequence, or the complete promoter sequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence may be compared to a reference sequence andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. Generally, the comparison windowis at least 20 contiguous nucleotides in length and optionally can be30, 40, 50, 100 or more contiguous nucleotides in length. Those of skillin the art understand that to avoid a high similarity to a referencesequence due to inclusion of gaps in the polynucleotide sequence, a gappenalty is typically introduced and is subtracted from the number ofmatches.

(c) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

(d) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%sequence identity, preferably at least 80%, more preferably at least 90%and most preferably at least 95%, compared to a reference sequence usingone of the alignment programs described using standard parameters.

Methods of aligning sequences for comparison are well known in the art.Gene comparisons can be determined by conducting BLAST (Basic LocalAlignment Search Tool; Altschul, et al., (1993) J. Mol. Biol.215:403-410; see also the BLAST page of the U.S. government's NationalCenter for Biotechnology Information, National Library of Medicine,National Institutes of Health) searches under default parameters foridentity to sequences contained in the BLAST “GENEMBL” database. Asequence can be analyzed for identity to all publicly available DNAsequences contained in the GENEMBL database using the BLASTN algorithmunder the default parameters.

For purposes of defining the present invention, GAP (Global AlignmentProgram) is used. GAP uses the algorithm of Needleman and Wunsch (J.Mol. Biol. 48:443-453, 1970) to find the alignment of two completesequences that maximizes the number of matches and minimizes the numberof gaps. Default gap creation penalty values and gap extension penaltyvalues in Version 10 of the Wisconsin Package® (Accelrys, Inc., SanDiego, Calif.) for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. Percent Similarity is the percent ofthe symbols that are similar. Symbols that are across from gaps areignored. A similarity is scored when the scoring matrix value for a pairof symbols is greater than or equal to 0.50, the similarity threshold.The scoring matrix used in Version 10 of the Wisconsin Package®(Accelrys, Inc., San Diego, Calif.) is BLOSUM62 (see, Henikoff andHenikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

Large amounts of the nucleic acids of the present invention may beproduced by replication in a suitable host cell. Natural or syntheticnucleic acid fragments coding for a desired fragment will beincorporated into recombinant nucleic acid constructs, usually DNAconstructs, capable of introduction into and replication in aprokaryotic or eukaryotic cell. Usually the nucleic acid constructs willbe suitable for replication in a unicellular host, such as yeast orbacteria, but may also be intended for introduction to (with and withoutintegration within the genome) cultured mammalian or plant or othereukaryotic cell lines. The purification of nucleic acids produced by themethods of the present invention is described, for example, in Sambrook,et al., Molecular Cloning. A Laboratory Manual, 2nd Ed. (Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1989) or Ausubel, et al.,Current Protocols in Molecular Biology, J. Wiley and Sons, NY (1992).

Nucleic acid constructs prepared for introduction into a prokaryotic oreukaryotic host may comprise a replication system recognized by thehost, including the intended nucleic acid fragment encoding the desiredprotein, and will preferably also include transcription andtranslational initiation regulatory sequences operably linked to theprotein encoding segment. Expression vectors may include, for example,an origin of replication or autonomously replicating sequence (ARS) andexpression control sequences, a promoter, an enhancer and necessaryprocessing information sites, such as ribosome-binding sites, RNA splicesites, polyadenylation sites, transcriptional terminator sequences, andmRNA stabilizing sequences. Secretion signals may also be included whereappropriate. Such vectors may be prepared by means of standardrecombinant techniques well known in the art and discussed, for example,in Sambrook, et al., Molecular Cloning. A Laboratory Manual, 2nd Ed.(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) orAusubel, et al., Current Protocols in Molecular Biology, J. Wiley andSons, NY (1992).

Vectors for introduction of genes both for recombination and forextrachromosomal maintenance are known in the art, and any suitablevector may be used. Methods for introducing DNA into cells such aselectroporation, calcium phosphate co-precipitation, and viraltransduction are known in the art, and the choice of method is withinthe competence of one skilled in the art (Robbins, Ed., Gene TherapyProtocols, Human Press, NJ (1997)).

Gene transfer systems known in the art may be useful in the practice ofthe present invention. These include viral and non-viral transfermethods. A number of viruses have been used as gene transfer vectors,including polyoma, i.e., SV40 (Madzak, et al., (1992) J. Gen. Virol.,73:1533-1536), adenovirus (Berkner, (1992) Curr. Top. Microbiol.Immunol. 158:39-61; Berkner, et al., (1988) Bio Techniques, 6:616-629;Gorziglia, et al., (1992) J. Virol. 66:4407-4412; Quantin, et al.,(1992) Proc. Natl. Acad. Sci. USA 89:2581-2584; Rosenfeld, et al.,(1992) Cell 68:143-155; Wilkinson, et al., (1992) Nucl. Acids Res.20:2233-2239; Stratford-Perricaudet, et al., (1990) Hum. Gene Ther.1:241-256), vaccinia virus (Mackett, et al., (1992) Biotechnology24:495499), adeno-associated virus (Muzyczka, (1992) Curr. Top.Microbiol. Immunol. 158:91-123; Ohi, et al., (1990) Gene 89:279-282),herpes viruses including HSV and EBV (Margolskee, (1992) Curr. Top.Microbiol. Immunol. 158:67-90; Johnson, et al., (1992) J. Virol.66:2952-2965; Fink, et al., (1992) Hum. Gene Ther. 3:11-19; Breakfield,et al., (1987) Mol. Neurobiol. 1:337-371; Fresse, et al., (1990)Biochem. Pharmacol. 40:2189-2199), and retroviruses of avian(Brandyopadhyay, et al., (1984) Mol. Cell Biol. 4:749-754; Petropouplos,et al., (1992) J. Virol. 66:3391-3397), murine (Miller, (1992) Curr.Top. Microbiol. Immunol. 158:1-24; Miller, et al., (1985) Mol. CellBiol. 5:431-437; Sorge, et al., (1984) Mol. Cell Biol. 4:1730-1737;Mann, et al., (1985) J. Virol. 54:401-407), and human origin (Page, etal., (1990) J. Virol. 64:5370-5276; Buchschalcher, et al., (1992) J.Virol. 66:2731-2739).

Non-viral gene transfer methods known in the art include chemicaltechniques such as calcium phosphate coprecipitation (Graham, et al.,(1973) Virology 52:456-467; Pellicer, et al., (1980) Science209:1414-1422), mechanical techniques, for example microinjection(Anderson, et al., (1980) Proc. Natl. Acad. Sci. USA, 77:5399-5403;Gordon, et al., (1980) Proc. Natl. Acad. Sci. USA, 77:7380-7384;Brinster, et al., (1981) Cell 27:223-231; Constantini, et al., (1981)Nature 294:92-94), membrane fusion-mediated transfer via liposomes(Felgner, et al., (1987) Proc. Natl. Acad. Sci. USA 84:7413-7417; Wang,et al., (1989) Biochemistry 28:9508-9514; Kaneda, et al., (1989) J.Biol. Chem. 264:12126-12129; Stewart, et al., (1992) Hum. Gene Ther.3:267-275; Nabel, et al., (1990) Science 249:1285-1288; Lim, et al.,(1992) Circulation 83:2007-2011), and direct DNA uptake andreceptor-mediated DNA transfer (Wolff, et al., (1990) Science247:1465-1468; Wu, et al., (1991) BioTechniques 11:474-485; Zenke, etal., (1990) Proc. Natl. Acad. Sci. USA 87:3655-3659; Wu, et al., (1989)J. Biol. Chem. 264:16985-16987; Wolff, et al., (1991) BioTechniques11:474485; Wagner, et al., 1990; Wagner, et al., (1991) Proc. Natl.Acad. Sci. USA 88:42554259; Cotton, et al., (1990) Proc. Natl. Acad.Sci. USA 87:4033-4037; Curiel, et al., (1991) Proc. Natl. Acad. Sci. USA88:8850-8854; Curiel, et al., (1991) Hum. Gene Ther. 3:147-154).

One skilled in the art readily appreciates that the methods describedherein are applicable to other species not specifically exemplified,including both plants and other non-human organisms. The followingexamples are intended to illustrate but not limit the invention.

EXAMPLE 1 Promoter Hairpin RNA Expression Affects Plant Fertility

This example demonstrates that the fertility or fertility potential ofplants can be altered by expression of hairpin RNA (hpRNA) moleculesspecific for the promoters of genes that encode proteins involved inmale fertility pathways.

Promoter hpRNA constructs were generated by linking a ubiquitin promoterto an inverted repeat of the desired promoter, including a NOS promotersegment between the inverted repeat sequences. Expression of eachconstruct generated a hpRNA specific for one of the following promoters:MS45, 5126, BS7, SB200, and PG47. Nucleic acid molecules and methods forpreparing the constructs and transforming maize were as previouslydescribed (Cigan, et al., (2001) Sex Plant Reprod. 14:135-142). Progeny(T1 generation) of transformed (T0) plants were analyzed.

Of 32 transformation events comprising hpRNA specific for the MS45 genepromoter, 29 produced T1 plants that were male sterile.

Of 32 transformation events comprising hpRNA specific for the 5126 genepromoter, 29 produced T1 plants that were male sterile.

Of 32 transformation events comprising hpRNA specific for the BS7 genepromoter, 23 produced T1 plants that either produced a small amount ofnon-viable pollen (“breaker” phenotype) or were male fertile butproduced only a small amount of viable pollen (“shedder” phenotype).

Of 31 transformation events comprising hpRNA specific for the SB200 genepromoter, 13 produced T1 plants of either the breaker or shedderphenotype.

Of 24 transformation events comprising hpRNA specific for the PG47 genepromoter linked to a construct for herbicide resistance, 15 revealed notransmission of herbicide resistance to the T1 seedling when usingpollen from the primary transformants. This is consistent with expectedpost-meiotic expression of PG47.

Anther RNA from plants expressing the various hpRNAs was analyzed bynorthern blot. For each target, six independent events were analyzed inthe T1 generation to determine whether hpRNA expression reduced steadystate RNA levels of the targeted genes. Anthers were staged at tetradrelease to early uninucleate stage of microspore development. Poly A⁺RNA was isolated, separated by electrophoresis, transferred tomembranes, and hybridized sequentially with probes specific for MS45,5126, BS7, SB200, NOS, and actin (RNA loading control). No MS45, 5126,or BS7 transcripts were detected in plants expressing hpRNA specific forthese endogenous promoters. Only a slight reduction of SB200 RNA wasobserved in plants expressing SB200 hpRNA.

Protein immunoblot analysis of anther proteins also was performedessentially as described previously (Cigan, et al., (2001) Sex PlantReprod. 14:135-142). For each target, six independent events wereanalyzed in the T1 generation to determine whether expression of thepromoter hpRNA reduced steady-state protein levels of the targetedgenes. Anthers were staged as above, ground in Laemelli buffer,separated by electrophoresis, and reacted sequentially with antibodiesspecific for MS45, BS7, SB200 or 5126 protein. Similar to the northernblot results, no MS45, 5126, or BS7 proteins were detected in plantsexpressing hpRNA specific for these endogenous promoters, and only aslight reduction of SB200 protein was observed for events comprisinghpSB200.

These results demonstrate that expression of promoter hpRNA canselectively suppress endogenous gene expression in plant cells. Inaddition, the results demonstrate that suppression of different genesinvolved in male sterility of plants can variously affect the plantphenotype, including the degree of male fertility.

EXAMPLE 2 Expression of Exogenous MS45 Gene Product Restores Fertility

This example demonstrates that plants rendered male-sterile byexpression of an MS45 promoter hairpin construct can be restored tofertility by expression of an exogenous MS45 gene construct.

Constructs were prepared containing the MS45 coding sequence operablylinked to a heterologous ubiquitin (UBI), 5126, SB200 or BS7 promoter;these constructs were introduced into ms45 ms45 plant cells. Regeneratedplants and their progeny were fertile, demonstrating that the nativepromoter of MS45 can be replaced with either a constitutive oranther-preferred promoter to confer a male-fertile phenotype to mutantms45 maize. (See also, Cigan, et al., (2001) Sex Plant Reprod.14:135-142).

Further, plants containing the UBI:MS45 or 5126:MS45 construct werecrossed to plants that were male sterile due to expression of an MS45gene promoter hpRNA. Progeny were tested by PCR for presence of the hpconstruct and either UBI:MS45 or 5126:MS45. RNA hybridization analysiswas conducted and fertility phenotypes were scored.

Northern blot analysis of RNA obtained from leaves of the progeny plantsrevealed that MS45 was expressed from the ubiquitin promoter in 7 of 12hp-containing progeny obtained from the UBI:MS45 cross. Further,expression of MS45 from the UBI promoter correlated with observedfertility in the progeny plants. These results demonstrate indicate thatMS45 is expressed from the constitutive ubiquitin promoter, and thatconstitutive expression of the MS45 gene product confers male fertilityin the progeny plants.

Further, anther RNA from these MS45hp maize plants containing 5126:MS45,BS7:MS45, or UBI:MS45 was analyzed. Anthers were staged at tetradrelease to early uninucleate stage of microspore development, and polyA+ RNA was collected, electrophoresed and hybridized sequentially withprobes for MS45, SB200, and BS7. MS45 was expressed in anthers of themale-fertile progeny plants whether driven by the constitutive UBIpromoter or by the anther-specific 5126 or BS7 promoters, with timing ofanther collection likely affecting strength of the signal. No MS45 RNAwas observed in the male-sterile hairpin-only containing plants. Theseresults demonstrate that suppression of MS45 expression due to the MS45hpRNA can be overcome by expressing MS45 from a heterologous promoterthat drives expression at least in anther cells.

The promoter expressing the MS45 gene can be derived from a source otherthan maize, and can be, for example, any plant promoter capable oftranscribing MS45 such that expression of the transcription unit rendersplants male fertile. For example, the rice and Arabidopsis homologs ofthe maize MS45, 5126, BS7 and MS26 genes have been isolated andidentified. Overall there is significant similarity between the codingregions, with conservation of the intronic regions. Importantly, thecorresponding promoters of rice and maize are approximately 50 to 60%identical, suggesting that these promoters may function sufficiently inmaize tapetum to transcribe the MS45 gene. To test this, each of therice MS45, rice BS7, rice MS26 and Arabidopsis 5126 promoters was fusedto the maize MS45 coding region and tested for ability of the constructto confer fertility when transformed into ms45 ms45 mutants. Using thistest system, a high frequency of male fertile plants was observed forall four constructs.

In certain respects, it is advantageous to use non-maize promoters toexpress the MS45 gene. For example, where promoter hpRNAs from the samespecies reduce target gene function such that the plant is non-viable ornon-reproductive, a promoter from a different species can be used totranscriptionally express the complementing gene function (e.g., MS45),thus circumventing this potential problem. Moreover, hpRNA constructscan be generated to target the non-maize promoters to suppress MS45 geneexpression as a means to reduce or abolish function and render the plantmale sterile by targeting the non-maize promoter used in the MS45expression cassette. For example, an ms45 homozygous recessive plant maybe transformed with an MS45 rice promoter homolog driving expression ofthe MS45 gene (MS45r::MS45), rendering the plant male fertile. Tosuppress expression of this MS45r::MS45 cassette, a second maize plantcan be generated which is heterozygous for the maize MS45 mutation andexpresses an MS45r promoter hpRNA. As there are no equivalent endogenousMS45 rice promoter target sequences in this maize plant, this plantwould be male fertile. This second plant can be crossed onto thehomozygous ms45 plant containing the MS45r::MS45 construct, and progenyscreened for the MS45r::MS45 and the MS45r hpRNA constructs. In thissituation, MS45r::MS45 gene function is suppressed by the presence andexpression of the MS45rHP, resulting in a male-sterile plant.

Use of such constructs is supported by the finding that expression ofthe rice 5126 promoter hp in maize does not result in male sterileplants. This is in contrast to the results obtained using a maize 5126promoter hp (see, Example 1) and suggests that expression of the rice5126 promoter hairpin is incapable of suppressing the endogenous maize5126 gene.

Taken together, the present Examples demonstrate that endogenous plantfertility genes can be inactivated using hpRNA mediated suppression, andthat a fertile phenotype can be restored in phenotypically sterileplants.

EXAMPLE 3 Promoter Specific Hairpin RNA Suppresses Transmission ofTransgene Mediated Herbicide Resistance

This example demonstrates that pollen of plants hemizygous for aUBI:PG47 hairpin construct is non-viable as determined bynon-transmission of herbicide resistance to T1 outcrosses when aherbicide resistance gene is linked to the PG47 hairpin construct.

An hpRNA specific for the PG47 gene promoter comprising an invertedrepeat of the PG47 gene promoter driven by a ubiquitin promoter(UBI:PG47hp), linked to a 35S:PAT construct, was introduced into plantcells. Pollen from plants expressing the transgene, representing 24 low-or single-copy transformation events, was carried to ears of wild-typemaize plants. Seed set on the ears was very good, and comparable to thatobserved when wild-type pollen was used. For each event, 32 seeds wereplanted in soil, and seedlings were sprayed 5 days post-germination with2× LIBERTY herbicide to detect transmission of UBI:PG47hp linked to35S:PAT.

It was expected that if PG47-specific hpRNA functioned at thepost-meiotic division of microspores, then viability would be normal,and 50% of the pollen would carry the transgene, providing herbicideresistance in 50% of the progeny. However, if PG47 function is requiredfor pollen viability, and the hairpin construct can suppress expressionof the PG47 gene product, then 50% of the pollen grains would benon-viable; all viable pollen would lack the transgene and be incapableof transmitting herbicide resistance. Non-functioning UBI:PG47hpconstructs would be detectable by the presence of herbicide resistantplants.

Fifteen of 24 events tested were herbicide sensitive. This resultdemonstrates that the UBI:PG47hp constructs suppress PG47 geneexpression in pollen, rendering 50% of the pollen non-viable andpreventing transmission of herbicide resistance operably linked to thesuppression construct.

EXAMPLE 4 Plants Containing Multiple Promoter Specific Hairpin RNAsSuppress Multiple Target Promoters

Plants containing 5126HP (i.e., a transgene encoding a 5126 promoterhpRNA) are used as pollen recipients for pollen from BS7HP expressingplants. In plants containing both 5126HP and BS7HP, endogenousexpression of 5126 and BS7 is suppressed, leading to a strongersterility phenotype than observed with either construct alone. Plantsare selected to contain either the 5126HP or BS7HP or both and advancedto maturity, and the fertility phenotypes of these resultant plants aredetermined.

Alternatively or in addition to crossing as a means to combine hairpinconstructs, one of said constructs, for example the 5126HP, can beplaced under the transcriptional control of an inducible promoter. Inthe absence of induction, these BS7HP-containing plants are capable ofproducing enough pollen to self. However, upon induction of the 5126HP,these plants are male sterile, and can be used as females during hybridproduction. This process depends upon the combined expression of thehairpin constructs (HPs) to render a plant infertile, while expressionof only one of the HPs does not impart sterility.

In certain embodiments, expression of both hpRNAs can be placed underthe transcriptional control of a single promoter. In this scenario, thehpRNAs can be designed to contain multiple target promoters within thesame encoded RNA. For example, the 5126 promoter region can bejuxtaposed to the BS7 promoter region and placed under thetranscriptional control of a single ubiquitin promoter or otherconstitutive, developmental or tissue preferred promoter, resulting inthe expression of an RNA containing a 5126 and BS7 hybrid hairpin thatdirects the suppression of both 5126 and BS7 endogenous genes. Anycombination and number of various promoters that target multiple anddifferent promoters can be used in the scheme. For example, a promoterthat regulates plant height genes and a promoter important to areproductive process can be combined, resulting in sterile plants havingshort stature.

EXAMPLE 5 Inbred Maintenance and Hybrid Production of Plants ContainingPromoter-Specific Hairpin RNAs Suppressing Target Promoters andComplementation Constructs

This example demonstrates how an inbred plant containing two constructs,a dominant hairpin RNA (hpRNA) construct specific for a promoter and anMS45 gene expressed from a tissue specific promoter, can be maintainedand used in the production of male sterile females for hybridproduction.

Inbred plants A1 and A2 are both homozygous recessive ms45 ms45.Fertility is restored to inbred A1 plants by introduction of a transgeneexpressing the MS45 coding region using the 5126 promoter. The A1 inbredplants also contain a BS7HP expressing construct. These plants can beselfed and maintained independently of inbred A2. In inbred A2 plants,fertility is restored by expressing the MS45 coding region using the BS7promoter. The A2 inbred plants also contain a 5126HP expressingconstruct. These plants can be selfed and maintained independently ofinbred A1 plants.

To generate seed for female inbreds for hybrid production, inbred A1 isdetasseled and fertilized using pollen from inbred A2. The resultantseed from this cross is planted and all of the progeny plants are malesterile due to the presence of the homozygous ms45 alleles and the5126HP and BS7HPs suppressing the fertility restoration genes, 5126-MS45and BS7-MS45, respectively. These plants are used as females in hybridproduction and pollinated with plants having wild-type MS45 generesulting in hybrid F1 seed. All plants derived from this seed areheterozygous for the MS45 gene and are therefore male fertile.

This example demonstrates that plants containing both dominantsuppression and restoring constructs can be maintained and used in ahybrid seed production strategy to generate sterile female inbreds andfertile hybrid plants.

EXAMPLE 6 Utility of Plants Containing Promoter Specific Hairpin RNAsSuppressing Target Pollen-Specific Promoters and Ms45 ComplementationConstructs for Hybrid Production and Inbred Maintenance

This example demonstrates how a method comprising the use of twoconstructs, a dominant hairpin RNA (hpRNA) construct specific for apollen-specific promoter and a restoring transgene, allows for thepropagation of a plant having a homozygous recessive reproductive traitwithout losing the homozygous recessive condition in the resultingprogeny, for use in the production of sterile plants for hybridproduction. This is accomplished by introducing into a plant at leastone restoring transgene construct, operably linking a first nucleotidesequence comprising a functional copy of a gene that complements themutant phenotypic trait produced by the homozygous recessive conditionwith a second functional nucleotide sequence which interferes with theformation, function, or dispersal of the male gametes of the plant. Thisconstruct is maintained in the hemizygous state and a plant containingsuch a construct is referred to as a maintainer. Interference with theformation, function, or dispersal of the male gamete may be accomplishedby linking the sequences interfering with formation, function, ordispersal of the male gamete with a gamete-tissue-preferred promoter.Since the transgene is in the hemizygous state, only half of the pollengrains produced contain the restoring transgene construct and none ofthese are viable due to the action of the second gene that prevents theformation of viable pollen. Therefore, when the maintainer plantcontaining such a linked construct is used as a pollen donor tofertilize the homozygous recessive plant, the only viable male gametesprovided to the homozygous recessive plant are those which contain therecessive allele, but do not contain any component of the transgeneconstruct. The progeny resulting from such a sexual cross are nontransgenic with respect to this transgene construct.

While no viable pollen produced by the maintainer contains the restoringtransgene construct, 50% of the ovules (the female gamete) will containthe restoring transgene construct. Therefore, the maintainer can bepropagated by self-fertilization, with the restoring transgene constructsegregating such that it will be contained in 50% of the seed of a selffertilized maintainer. By linking the restoring transgene construct witha selectable marker, the 50% of the seed containing the transgene can beisolated to propagate the maintainer population, which remainshomozygous for the recessive gene and hemizygous for the restoringtransgene construct. In this scenario, a single inbred can bemaintained.

Inbred A1 is homozygous recessive for the fertility gene ms45. Inbred A1plants contain a construct in which male fertility is restored byexpressing the MS45 coding region using a tissue specific promoter, forexample the native MS45 promoter. Inbred A1 plants also contain ahairpin construct targeted to suppress a pollen expressed promoter, inthis example, a PG47HP expressing construct operably linked to the MS45restoring construct; and a selectable or screenable marker, for example,a marker that confers herbicide resistance and/or a construct thatserves as a visual or detectable marker for plant and/or seed screening.These plants are fertile and can be selfed and maintained. The seed onthese plants will segregate 50:50 for the transgene because onlynon-transgenic pollen is viable and capable of effecting fertilizationof an ovule, 50% of which contain the construct.

To generate seed for female inbreds for hybrid production, in one row,only non-transgenic plants from inbred A1 are maintained; these plantsare homozygous recessive ms45 and male sterile. In an adjacent row, bothtransgenic and non-transgenic plants from inbred A1 are grown. Fertilityin this row segregates one to one (fertile to sterile); fertile plantsare used to pollinate the sterile plants in the adjacent row. The seedfrom this cross is non-transgenic for the operably linked restorer, thehpRNA and the screenable marker constructs, and all of the progeny aremale sterile due to the presence of the homozygous ms45 allele. Theseplants are used as females in hybrid production and pollinated withplants having wild-type MS45 gene resulting in hybrid F1 seed. Allplants derived from this seed are heterozygous for the MS45 gene and,therefore, male fertile.

This example demonstrates that plants containing a dominant pollensuppression hairpin construct and a fertility restoring construct can bemaintained as inbreds and used in a hybrid seed production strategy togenerate sterile female inbreds and fertile hybrid plants.

EXAMPLE 7 Combinations

Two or more construct components described herein may be combined invarious ways to create systems for controlling gene expression. Suchcombinations may be made by linking said components within a singlevector, by using multiple vectors in simultaneous or sequentialtransformations, and/or by breeding of plants comprising one or morecomponents. Possible components are described below and in Table 1.Table 2 provides representations of illustrative, but not exhaustive,combinations useful in controlling male fertility.

For example, the components may include promoters or coding regionsother than those listed, and the order of the components within theconstructs may be different than those shown. Further, a construct couldcomprise individual promoter/coding sequence combinations, or onepromoter driving transcription of multiple coding sequence components.As an example of the latter, a construct could comprise a constitutivepromoter driving transcription of an MS45 coding sequence as well as apolynucleotide encoding a gene product involved in producing orregulating a screenable marker (for example, pigment) to create a fusionproduct. This would allow screening for transformants using any tissueof the plant, while expression of the MS45 results in male fertility.

Within any of the constructs, one or more promoter hairpin componentscould be included, for example within an intron of any of the encodedgenes, or within a 5′ or 3′ non-coding region, or as an initial orterminal extension. A hairpin may target a single promoter, or two ormore promoters, within a single transcribed RNA. Pollen-promoter hairpinconfigurations, and/or polynucleotides encoding pollen-disruptingpolypeptides, can serve to prevent transgene transmission through themale gametes.

Pollen-preferred or pollen-specific promoters (“Poll-P”) include, forexample, PG47, P95 (onset between mid- and late-uninucleate stages; see,SEQ ID NO: 2), and P67 (profile similar to P95, more highly expressed atmid-uninucleate stage; see, SEQ ID NO: 1).

Tapetum-specific (“Tisp-P”) or tapetum-preferred (“Tap-P”) promotersinclude, for example, MS45 (U.S. Pat. No. 6,037,523); 5126 (U.S. Pat.No. 5,837,851); Bs7 (WO 02/063021); and SB200 (WO 02/26789).

Other tissue-specific or tissue-preferred promoters useful in theinvention include, for example, Br2 (Science 302(5642):71-2, 2003),CesA8, and LTP2 (Plant J 6:849-860, 1994).

Constitutive promoters (“ConstP”) include, for example, the CaMV 35Spromoter (WO 91/04036 and WO 84/02913); and the maize ubiquitinpromoter.

Male fertility genes (“MF”) useful in the invention include, forexample, MS45 (Cigan, et al., (2001) Sex. Plant Repro. 14:135-142; U.S.Pat. No. 5,478,369) and MS26 (US Patent Application Publication Number20030182689).

Pollen ablation genes (“Cytotox”) useful in the invention include DAM(GenBank J01600, Nucleic Acids Res. 11:837-851 (1983); alpha-amylase(GenBank L25805, Plant Physiol. 105(2):759-760 (1994)); D8 (Physiol.Plant. 100(3):550-560 (1997)); SacB (Plant Physiol. 110(2):355-363(1996)), lipases and ribonucleases. In this regard, a singlepolypeptide, or a fusion of two or more polypeptides to generate afusion product, is contemplated. Selectable marker systems useful in thepractice of the invention include, for example, herbicide resistanceconferred by PAT or MoPAT.

Screenable marker systems useful in the practice of the invention, forexample in identifying transgenic seed among progeny of a selfedmaintainer line, include GFP (Gerdes, (1996) FEBS Lett. 389:44-47;Chalfie, et al., (1994) Science 263:802), RFP, DSred (Dietrich, et al.,(2002) Biotechniques 2(2):286-293), KN1 (Smith, et al., (1995) Dev.Genetics 16(4):344-348), CRC, P, (Bruce, et al., (2000) Plant Cell12(1):65-79, and Sugary1 (Rahman, et al., (1998) Plant Physiol.117:425-435; James, et al., (1995) Plant Cell 7:417-429; U18908).

Hairpin configurations may comprise, for example, PG47hp, P95hp, or P67hp. A hairpin may target a single promoter or may target two or morepromoters by means of a single transcribed RNA. The hairpin could belocated in any appropriate position within the construct, such as withinan intron of any of the encoded genes or within 5′ or 3′ non-codingregions.

TABLE 1 Symbol Description Example Poll-P Pollen Promoter PG47, P95, P67Tisp-P Tissue Specific Promoter Br2, CesA8, LTP2 Tap-P Tapetum PromoterMs45, 5126, Bs7, Sb200 ConstP Constitutive Promoter 35S, Ubi MFFertility Gene Ms45, Ms26 Cytotox Cytotoxic Gene DAM, Alpha-Amylase, D8,SacB Herb R Herbicide Resistance PAT, MoPAT Screen Screenable MarkerRFP, GFP, KN1, CRC, Su1 HP Hairpin PG47hp, P95hp, P67hp

TABLE 2 Description Components Single cytotox + SelectionPoll-P:Cytotox/Tap-P:MF/ConstP:Herb R Single cytotox + Selection +Screen Poll-P:Cytotox/Tap-P:MF/ConstP:Herb R/Tisp-P:Screen Doublecytotox + Selection Poll-P:Cytotox/Poll-P:Cytotox/Tap-P:MF/ConstP:Herb RSingle cytotox + Screen Poll-P:Cytotox/Tap-P:MF/Tisp-P:Screen Doublecytotox + Screen Poll-P:Cytotox/Poll-P:Cytotox/Tap-P:MF/Tisp-P:ScreenHairpin + Single cytotox + SelectionConstP:HP/Poll-P:Cytotox/Tap-P:MF/ConstP:Herb R Hairpin + Singlecytotox + Screen ConstP:HP/Poll-P:Cytotox/Tap-P:MF/Tisp-P:ScreenHairpin + Selection ConstP:HP/Tap-P:MF/ConstP:Herb R Hairpin + ScreenConstP:HP/Tap-P:MF/Tisp-P:Screen Hairpin/Male fertile fusion + ScreenConstP:HP + MF/Tisp-P:Screen Hairpin/Male fertile fusion + SelectionConstP:HP + MF/ConstP:Herb R Embedded Hairpin/Male fertile + ConstP:MFEmbedded HP/ConstP:Herb R Selection Embedded Hairpin/Male fertile +ConstP:MF Embedded HP/Tisp-P:Screen Screen Embedded Hairpin/ScreenTap-P:MF/ConstP:Screen Embedded HP Single Cytotox EmbeddedPoll-P:Cytotox/Tap-P:MF/ConstP:Screen Embedded HP Hairpin/ScreenConstitutive Fertility/Screen with ConstP:(MF + Screen) Embedded HPEmbedded Hairpin Tap-P:Cytotox/ConstP:(MF + Screen) Embedded HP

EXAMPLE 8 Visual Marker-Based Selection

The experiments described below were designed to ask whether the maizep1 gene, when expressed from various non-p1 promoters, could be used asa visual marker for seed carrying a linked transgene. As part of theexperimental design, coloration of seed from the transformed plant, aswell as coloration of seed generated by outcrossing pollen from thetransformed plant, was tested to examine inheritance of maternal andpaternal p1 gene expression.

The p1 gene of maize is a Myb-related transcriptional activatordemonstrated to regulate the a1 and c2 genes to produce 3-deoxyflavonoids, such as C-glycosyl flavones, 3-deoxyanthocyanins,flavan-4-ols and phlobaphenes (Grotewold, et al., (1991) PNAS88:4587-4591). Synthesis of these and related compounds results in thecoloration of floral organs including pericarp, cob, silks, husks andtassel glumes (Cocciolone, et al., (2001) Plant J 27(5):467-478).Typically, expression of this gene is maternal; that is, outcrossing ofthe p1 gene does not confer coloration to reproductive parts until thenext generation is grown from seed. As the p1 gene has been shown toconfer color to non-reproductive maize tissues by constitutiveexpression in BMS (Black Mexican Sweet) cells (Grotewold, et al., PICell 1998), expression of the p1 gene was investigated by placing the p1gene under the transcriptional control of the maize seed-preferredpromoters END2 and LTP2. Constitutive promoters rice Actin and maizeUbiquitin were also used to transcriptionally regulate the p1 gene.These vectors would test whether expression of the p1 gene would confercolor differences sufficient for use as a visual marker.

The following vectors were introduced into maize by Agrobacteriumtransformation and tested for seed color of both the transformed plantand ears pollinated with pollen from the transformed plants.

23030 End2:P1-UbimoPAT 23066 Actin:P1-UBImoPat 23069 LTP2:P1-UBImoPat23528 End2:P1-35SPAT 23535 LTP2:P1-35S:PAT 23537 UBI:P1-35S:PAT

Transformation with PHP23030 and PHP23069 has produced plantsdemonstrating segregating colored seed both on ears of the primarytransformed plants and on ears pollinated by pollen from thesetransformed plants. For PHP23030, 12 of the 14 independent events usedfor outcrossing demonstrated brown colored kernels segregating among theyellow kernels at nearly a 1:1 segregation ratio. Ears on the primarytransformants were pollinated with pollen from non-transformed plantsand the kernels on these ears also segregated brown:yellow kernels atnearly a 1:1 ratio. Identical results were observed with three of thefour events generated with PHP23069.

Brown and yellow seed from 5 single-copy PHP23030 events were sorted andplanted to test for germination of the brown seed and co-segregation ofthe linked herbicide resistance marker, 35SPAT, with the coloredkernels. In this small test, the majority (>95%) of the brown seedproduced herbicide resistant plants, whereas the 39 of the 40 seedlingsgerminated from yellow seed were herbicide sensitive.

Close examination of the brown seed from PHP23030 revealed that thealeurone layer fluoresced green, while the endosperm of brown seed fromPHP23069 showed strong green fluorescence when compared to yellowsegregating seed derived from the same ear. This is consistent with theobservation of green fluorescence observed in BMS cells bombarded with35S:P1 (Grotewold, et al., (1998) Plant Cell 10(5):721-740). Moreover,examination of the transformed callus with PHP23528 (End2:P1-35SPAT) andPHP23535 (LTP2:P1-35S:PAT) revealed, in contrast to untransformed GS3callus, both PHP23528- and PHP23535-containing callus fluoresced green.The observation of green fluorescence in these transformed callus andthe co-segregation of brown kernels with the herbicide selectable markerin transformed plants indicates that expression of p1 from at leastseed-preferred promoters can be used as a visual marker to identifytransformed maize tissues.

EXAMPLE 9 Alternatives for Pollen Cytotoxicity

As shown in Tables 1 and 2, disruption of pollen function may beaccomplished by any of numerous methods, including targeted degradationof starch in the pollen grain or interference with starch accumulationin developing pollen. For example, a construct comprising thealpha-amylase coding region is operably linked to a pollen specificpromoter. The native secretory signal peptide region may be present; maybe removed; or may be replaced by an amyloplastid-targeted signalpeptide. In other embodiments, a construct may comprise apollen-specific promoter operably linked to a coding region forbeta-amylase; or for a debranching enzyme such as Sugary1 (Rahman, etal., (1998) Plant Physiol. 117:425-435; James, et al., (1995) Plant Cell7:417-429; U18908) or pullulanase (Dinges, et al., (2003) Plant Cell15(3):666-680; Wu, et al., (2002) Archives Biochem. Biophys.406(1):21-32).

For example, hairpin constructs are created which target the promoter ofthe maize Sugary1 gene. Due to loss of the starch debranching enzymeactivity, sugary1 mutants display shrunken kernels. Constitutiveexpression of the promoter inverted repeat should cause loss of Su1promoter activity and result in inherited altered kernel morphology.

Although the invention has been described with reference to the aboveexample, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the claims.

All publications and patent herein referred to are hereby incorporatedby reference to the same extent as if each was individually soincorporated.

1. A promoter providing preferential expression of an operably-linked polynucleotide in developing pollen, comprising the sequence of SEQ ID NO:
 1. 2. An expression cassette comprising the promoter of claim 1 and an operably-linked polynucleotide.
 3. A transformation vector comprising the expression cassette of claim
 2. 4. A plant stably transformed with the expression cassette of claim
 2. 5. The plant of claim 4, wherein the plant is a monocot.
 6. The plant of claim 5, wherein the monocot is maize, wheat, rice, barley, sorghum, or rye.
 7. A transformed plant cell comprising the expression cassette of claim
 2. 8. A method for selectively expressing a polynucleotide in a plant cell, the method comprising transforming a plant cell with the expression cassette of claim
 2. 9. The method of claim 8 further comprising regenerating a stably transformed plant from the transformed plant cell.
 10. The method of claim 9 wherein fertility of the transformed plant is impacted. 